Nonnucleoside inhibitors of reverse transcriptase, composite binding pocket and methods for use thereof

ABSTRACT

Novel compounds that are potent inhibitors of HIV reverse transcriptase (RT) are described in the invention. Thes novel compounds also inhibit replication of a retrovirus, such as human immunodeficiency virus-1 (HIV-1). The novel compounds of the invention include analogs and derivatives of phenethylthiazolylthiourea (PETT), of dihydroalkoxybenzyloxopyrimidine (DABO), and of 1-[( 2 -hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT). The invention additionally provides a composite HIV reverse-transcriptase (RT) nonnucleoside inhibitor (NNI) binding pocket constructed from a composite of multiple NNI-RT complexes The composite RT-NNI binding pocket provides a unique and useful tool for designing and identifying novel, potent inhibitors of reverse transcriptase.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The inventors acknowledge and appreciate the assistance of Dr. EliseSudbeck.

BACKGROUND OF THE INVENTION

Design of potent inhibitors of human immunodeficiency virus (HIV-1)reverse transcriptase (RT), an enzyme responsible for the reversetranscription of the retroviral RNA to proviral DNA, has been a focalpoint in translational AIDS research efforts (Greene, W. C., New EnglandJournal of Medicine, 1991, 324, 308-317; Mitsuya, H. et al., Science,1990, 249, 1533-1544; De Clercq, E., J. Acquired Immune Defic. Syndr.Res. Human. Retrovirus, 1992, 8, 119-134). Promising inhibitors includenonnucleoside inhibitors (NNI), which bind to a specific allosteric siteof HIV-1 RT near the polymerase site and interfere with reversetranscription by altering either the conformation or mobility of RT,thereby leading to noncompetitive inhibition of the enzyme (Kohlstaedt,L. A. et al., Science, 1992, 256, 1783-1790).

NNI of HIV-1 RT include the following:

-   (a) 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymines (HEPT;    Tanaka, H. et al., J. Med. Chem., 1991, 34, 349-357; Pontikis, R. et    al., J. Med. Chem., 1997, 40, 1845-1854; Danel, K., et al., J. Med.    Chem., 1996, 39, 2427-2431; Baba, M., et al., Antiviral Res, 1992,    17, 245-264);-   (b) tetrahydroimidazobenzodiazepinethiones (TIBO; Pauwels, R. et    al., Nature, 1990, 343, 470-474);-   (c) bis(heteroaryl)piperazines (BHAP; Romero, D. L. et al., J. Med.    Chem., 1993, 36, 1505-1508);-   (d) dihydroalkoxybenzyloxopyrimidine (DABO; Danel, K. et al., Acta    Chemica Scandinavica, 1997, 51, 426-430; Mai, A. et al., J. Med.    Chem., 1997, 40, 1447-1454);-   (e) 2′-5′-bis-O-(tertbutyldimethylsilyl)-3′-spiro-5″-(4″-amino-1″,    2″-oxathiole-2″, 2″-dioxide) pyrimidines (TSAO; Balzarini, J. et    al., Proc. Natl. Acad. Sci. USA, 1992, 89, 4392-4396); and-   (f) phenethylthiazolylthiourea (PETT) derivatives (Bell, F. W. et    al., J. Med. Chem., 1995, 38, 4929-4936; Cantrell, A. S. et al., J.    Med. Chem., 1996, 39, 4261-4274).

Current protein structure-based drug design efforts rely heavily oncrystal structure information of the target binding site. A number ofcrystal structures of RT complexed with NNIs (including α-APA, TIBO,Nevirapine, BHAP and HEPT derivatives) have been reported, and suchstructural information provides the basis for further derivatization ofNNI aimed at maximizing binding affinity to RT. However, the number ofavailable crystal structures of RT NNI complexes is limited, and nostructural information has been reported for RT-PETT complexes orRT-DABO complexes. Given the lack of structural information, researchersmust rely on other design procedures for preparing active PETT and DABOderivatives. One of the first reported strategies for systematicsynthesis of PETT derivatives was the analysis of structure-activityrelationships independent of the structural properties of RT and led tothe development of some PETT derivatives with significant anti-HIVactivity (Bell, F. W. et al., J. Med. Chem., 1995, 38, 4929-4936;Cantrell, A. S. et al., J. Med. Chem., 1996, 39, 4261-4274). Theinclusion of structural information in the drug design process shouldlead to more efficient identification of promising RT inhibitors.

Although the crystal structure of an RT-NNI complex can be used toprovide useful information for the design of a different type of NNI,its application is limited. For example, an analysis of the RT-APA(α-anilinophenylacetamide) complex structure would not predict that thechemically dissimilar inhibitor TNK (6-benzyl-1-benzyloxymethyl uracil)could bind in the same region. The RT-APA structure reveals that therewould not be enough room in the APA binding site for the1-benzyloxymethyl group of TNK (Hopkins, A. L. et al., J. Med. Chem.,1996, 39, 1589-1600). Nevertheless TNK is known to bind in this regionas evidenced by the crystal structure of RT-TNK which shows that RTresidues can adjust to accommodate the 1-benzyloxymethyl group.Conversely, an analysis of the RT-TNK complex would not predictfavorable binding of APA in the TNK binding site. The structure does notshow how residue E138 can move to accommodate the 2-acetyl group of theo-APA inhibitor.

Thus, any NNI binding pocket model based on an individual RT-NNI crystalstructure would have limited potential for predicting the binding ofnew, chemically distinct inhibitors. To overcome this problem, theinvention disclosed herein uses the NNI binding site coordinates ofmultiple, varied RT-NNI structures to generate a composite molecularsurface. A specific embodiment of the invention is a composite molecularsurface or binding pocket generated from nine distinct RT-NNI complexes,and reveals a larger than presumed NNI binding pocket not shown orpredicted by any of the individual structures alone (FIG. 2A). Thisnovel composite binding pocket, together with a computer dockingprocedure and a structure-based semi-empirical score function, providesa guide to predict the energetically favorable position of novel PETT,DABO, and HEPT derivatives, as well as other novel compounds, in the NNIbinding site of RT.

The invention further provides a number of computational tools which setforth a cogent explanation for the previously unexplained and notunderstood relative activity differences among NNIs, including PETT,DABO, and HEPT derivatives, and reveals several potential ligandderivatization sites for generating new active derivatives. Disclosedherein is the structure-based design of novel HEPT derivatives and thedesign and testing of non-cytotoxic PETT and DABO derivatives whichabrogate HIV replication in human peripheral blood mononuclear cells atnanomolar concentrations with an unprecedented selectivity index of>10⁵.

One procedure useful in structure-based rational drug design is docking(reviewed in Blaney, J. M. and Dixon, J. S., Perspectives in DrugDiscovery and Design, 1993, 1, 301). Docking provides a means for usingcomputational tools and available structural data on macromolecules toobtain new information about binding sites and molecular interactions.Docking is the placement of a putative ligand in an appropriateconfiguration for interacting with a receptor. Docking can beaccomplished by geometric matching of a ligand and its receptor, or byminimizing the energy of interaction. Geometric matching is faster andcan be based on descriptors or on fragments.

Structure-based drug design efforts often encounter difficulties inobtaining the crystal structure of the target and predicting the bindingmodes for new compounds. The difficulties in translating the structuralinformation gained from X-ray crystallography into a useful guide fordrug synthesis calls for continued effort in the development ofcomputational tools. While qualitative assessments of RT-inhibitorcomplexes provide helpful information, systematic quantitativeprediction of inhibitory activity of new compounds based on structuralinformation remains a challenge.

There is a need for more complete information on the structure andflexibility of the NNI binding pocket and for an improved model of thebinding pocket to serve as a basis for rational drug design. Inaddition, there is a need for more effective inhibitors of reversetranscriptase, particularly HIV-1 reverse transcriptase.

The invention disclosed herein addresses these needs by providing amodel for the three-dimensional structure of the RT-NNI binding pocketbased on the available backbone structure of RT-DNA complex and fullstructure of RT complexed with several NNI compounds. Structuralinformation from multiple RT-NNI complexes was combined to provide asuitable working model. In one embodiment, the NNI binding sitecoordinates of nine RT-NNI structures is used to generate a compositemolecular surface revealing a larger than presumed NNI binding pocket.This pocket, together with docking and a structure-based semi-empiricalscore function, can be used as a guide for the synthesis and analyses ofstructure-activity relationships for new NNI of RT, including newderivatives of HEPT, DABO, and PETT, as well as novel compounds havinglittle or no relationship to known NNIs. The practical utility of thisnovel composite model is illustrated and validated by the observedsuperior potency of new PETT and S-DABO derivatives as anti-HIV agents,described herein.

SUMMARY OF THE INVENTION

The invention provides novel compounds which inhibit reversetranscriptase (RT) and which inhibit replication of a retrovirus, suchas human immunodeficiency virus-1 (HIV-1). In one embodiment, the novelcompounds of the invention are analogs or derivatives ofphenethylthiazolylthiourea (PETT), dihydroalkoxybenzyloxopyrimidine(DABO) or 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT).Alternatively, the novel compounds of the invention bind the NNI bindingpocket, but are not related to any known NNI. Specific compounds of theinvention are described more fully in the Detailed Description and inthe Examples below.

The invention additionally provides compositions and methods forinhibiting reverse transcriptase activity of a retrovirus, such asHIV-1, by contacting the RT binding site of the retrovirus with acompound of the invention. The methods of the invention are useful forinhibiting replication of a retrovirus, such as HIV-1 and includetreating a retroviral infection in a subject, such as an HIV-1infection, by administering a compound or composition of the invention,for example, in a pharmaceutical composition.

The invention further provides a composite ligand binding pocketconstructed by superimposing multiple structures of ligand-binding sitecomplexes. Preferably, the composite binding pocket is constructed bysuperimposing the structures of at least one each of the following NNIcomplexed with RT: a compound, analog or derivative of HEPT or MKC; TNK,APA, Nevipapine, and TIBO. In one embodiment, the composite ligandbinding pocket is an HIV-1 reverse-transcriptase (RT) nonnucleosideinhibitor (NNI) binding pocket constructed by superimposing ninestructures of NNI-RT complexes, preferably having the coordinates setforth in Table 9.

Using the novel composite binding pocket of the invention, compoundsthat bind to the NNI binding site of reverse transcriptase can beidentified and/or screened. For example, a useful inhibitor isidentified by analyzing the fit of a candidate compound to the compositebinding pocket is analyzed. In one embodiment, the comparing comprisesanalyzing the molecular surface of the composite binding pocket. Theextent of contact between the molecular surface of the compound and themolecular surface of the binding pocket can be visualized, and any gapspace between the compound and the composite binding pocket can bedetermined and quantified. The candidate inhibitory compound can bedocked in the composite binding pocket, and its binding characteristicsanalyzed. For example, an estimate of the inhibition constant for thedocked compound can be calculated. The value of the inhibition constantis inversely related to the affinity of the candidate compound for thebinding pocket.

Using information provided by the composite binding pocket of theinvention, novel inhibitors of reverse transcriptase can be designed andscreened. Using molecular modeling techniques, a compound can be dockedinto an RT-NNI binding pocket, and the complex analyzed for its bindingcharacteristics. Gap space or regions that do not demonstrate optimumclose contacts between the compound and the binding pocket areidentified, permitting the compound to be modified to better occupy thesite. In such a method, novel inhibitors of reverse transcriptase aredesigned and screened.

Also provided by the invention are inhibitors of reverse transcriptaseidentified or designed by analyzing the compound's structural fit to thebinding pocket. Potent inhibitors designed and confirmed using thecomposite binding pocket of the invention include analogs andderivatives of known NNI, such as phenethylthiazolylthiourea (PETT)analogs, dihydroalkoxybenzyloxopyrimidine (DABO) analogs, and1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) analogs.

The compounds of the invention may be combined with carriers and/oragents to enhance delivery to sites of viral infection, such astargeting antibodies, cytokines, or ligands. The compounds may includechemical modifications to enhance entry into cells, or may beencapsulated in various known delivery systems.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a model of the HIV-1 reverse transcriptase (RT) active site,derived primarily from two crystal structures: HIV-1 RT (PDB access codehni) and HIV-1 RT with DNA fragment (PDB access code hmi). The bindingsite for non-nucleoside inhibitors is labeled NNI. The site fornucleoside inhibitors is labeled dNTP which includes the 3′ terminus ofDNA. Features describing the geometry of the binding region include thethumb, palm, fingers, and hinge region of RT.

FIG. 1B shows models of compound I-3 (color coded by atom type) andcompound I-4 (in blue) in NNI binding site of HIV reverse transcriptase,positioned by docking procedure. Wing 1 and Wing 2 represent twodifferent regions of the NNI binding site.

FIG. 2A shows a composite binding pocket of NNI active site of HIV-1 RT.Grid lines represent the collective van der Waals surface of ninedifferent inhibitor crystal structures superimposed in the active siteand highlight the available space for binding (inhibitor structuresinclude HEPT, MKC, TNK, APA, Nevirapine, N-ethyl Nevirapine derivative,8-Cl TIBO, and two 9-Cl TIBO compounds, with PDB access codes rti, rt1,rt2, hni, vrt, rth, hnv, rev and tvr, respectively). The surface iscolor-coded for hydrogen bonding (red), hydrophobic (gray) andhydrophilic (blue) groups of the superimposed inhibitors. The hydrogenatoms were not included.

FIG. 2B shows a composite binding pocket (purple) superimposed on theactive site residues of RT taken from the crystal structure coordinatesof RT complexed with 8-Cl-TIBO (pdb access code: hnv). In the compositebinding pocket, there are a number of regions which are larger thanthose defined by residues in individual crystal structures. Residuesshown here which extend past the purple surface and toward the center ofthe binding site represent regions which are considered flexible andcould be displaced by an appropriate inhibitor.

FIG. 3A shows a model of compound trouvirdine docked in the NNI bindingsite and color-coded by atom type. Spheres represent the sites of themolecular surface which are in contact with protein residues and areunavailable for future modification.

FIG. 3B shows a model of PETT compound I-3 docked in the NNI bindingsite and color-coded by atom type. Spheres represent the sites of themolecular surface which are in contact with protein residues and areunavailable for future modification.

FIG. 4A shows a stereo model of compound I-2 and grid shown in red whichrepresents gaps between the compound and protein residues (each redline=1 Å distance). Dashed lines show the nearest distance between anatom in the compound and the gap net which does not intersect thespheres shown in FIG. 3A.

FIG. 4B shows a stereo model of PETT compound I-3 and grid shown in redwhich represents gaps between the compound and protein residues (eachred line=1 Å distance). Dashed lines show the nearest distance betweenan atom in the compound and the gap net which does not intersect thespheres shown in FIG. 3B.

FIG. 5A shows a stereoview of compound trovirdine in the compositebinding pocket which was constructed from combined coordinates of RTcomplexed with nine different NNI compounds.

FIG. 5B shows a stereoview of PETT compounds I-3 (in magenta) and I-4(multicolor) in the composite binding pocket which was constructed fromcombined coordinates of RT complexed with nine different NNI compounds.

FIG. 6 shows a model of PETT compound II-4 docked in the NNI bindingsite and color-coded by atom type, as described above for FIG. 3A. Thesurface of the composite binding pocket is color-coded for hydrogenbonding (red), hydrophobic (gray) and hydrophilic (blue) groups of thesuperimposed inhibitors.

FIG. 7A is a view of the composite binding pocket of the NNI active siteof HIV-1 RT. The DABO compound 3c is superimposed in the NNI compositebinding site of the crystal structure of the RT/MKC-442 complex(hydrogen atoms not shown). MKC-442 (from crystal structure) is shown inpink, and compound 3c (from docking calculations) in multicolor.Compound 3c was docked into the active site of the RT/MKC complex (PDBaccess code: rt1) and then superimposed into the NNI composite bindingpocket based on the matrix used in the pocket construction. The S2substituent of the DABO compound 3c occupies the same region of thebinding pocket as the N1 substituent of the HEPT derivative MKC-442.

FIG. 7B is a view of the composite binding pocket of the NNI active siteof HIV-1 RT. An X-ray crystal structure of DABO compound 3b issuperimposed on the docked model of DABO compound 3d in the NNIcomposite binding pocket of RT, demonstrating their remarkably similarconformations.

FIG. 8 is an ORTEP drawing of the room temperature X-ray crystalstructure of DABO compound 3b (30% ellipsoids).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, a “retrovirus” includes any virus that expresses reversetranscriptase. Examples of a retrovirus include, but are not limited to,HIV-1, HIV-2, HTLV-I, HTLV-II, FeLV, FIV, SIV, AMV, MMTV, and MoMuLV.

As used herein, “reverse transcriptase (RT)” refers to an enzyme havingan NNI binding site similar to that of HIV-1 RT and to which ligandswhich bind the composite binding pocket of the invention bind.

As used herein, “reverse transcriptase (RT) activity” means the abilityto effect reverse transcription of retroviral RNA to proviral DNA. Onemeans by which RT activity can be determined is by measuring viralreplication. One measure of HIV-1 viral replication is the p24 coreantigen enzyme immunoassay, for example, using the assay commerciallyavailable from Coulter Corporation/Immunotech, Inc. (Westbrooke, Mich.).Another means by which RT activity is analyzed is by assay ofrecombinant HIV-1 reverse transcriptase (rRT) activity, for example,using the Quan-T-RT assay system commercially available from Amersham(Arlington Heights, Ill.) and described in Bosworth, et al., Nature1989, 341: 167-168.

As used herein, a compound that “inhibits replication of humanimmunodeficiency virus (HIV)” means a compound that, when contacted withHIV-1, for example, via HIV-infected cells, effects a reduction in theamount of HIV-1 as compared with untreated control. Inhibition ofreplication of HIV-1 can be measured by various means known in the art,for example, the p24 assay disclosed herein.

As used herein, a “nonnucleoside inhibitor (NNI)” of HIVreverse-transcriptase (HIV-RT) means a compound which binds to anallosteric site of HIV-RT, leading to noncompetitive inhibition ofHIV-RT activity. Examples of nonnucleoside inhibitors of HIV-RT include,but are not limited to, tetrahydroimidazobenzodiazepinthiones (TIBO),1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymines (HEPT),bis(heteroaryl)piperazines (BHAP),2′-5′-bis-O-(tertbutyldimethylsilyl)-3′-spiro-5″-(4″-amino-1″,2″-oxathiole-2″, 2″-dioxide) pyrimidines (TSAO),dihydroalkoxybenzyloxopyrimidine (DABO) and phenethylthiazolylthiourea(PETT) analogs. In one embodiment of the invention, the nonnucleosideinhibitor of HIV-RT is a PETT analog. In another embodiment of theinvention, the nonnucleoside inhibitor of HIV-RT is a DABO analog. Inanother embodiment of the invention, the nonnucleoside inhibitor ofHIV-RT is a HEPT analog.

As used herein, a “composite FIV reverse-transcriptase (RT)nonnucleoside inhibitor (NNI) binding pocket” or “composite bindingpocket” means a model of the three-dimensional structure of a ligandbinding site, such as the nonnucleoside inhibitor binding site of HIV-RTconstructed from a composite of multiple ligand-binding site complexes.The composite binding pocket represents a composite molecular surfacewhich reveals regions of flexibility within the binding site. Flexibleresidues within the NNI binding site include Tyr180, Tyr181, Tyr318,Tyr319, Phe227, Leu234, Trp229, Pro95, and Glu138 (the latter from thep51 subunit of RT). Examples of such a model include, but are notlimited to, a composite molecular surface developed with the aid ofcomputer software and based on a composite of coordinates of multipleRT-NNI complexes, as disclosed herein. In one embodiment, the bindingpocket has the coordinates set forth in Table 9.

As used herein, a “compound that fits the nonnucleoside inhibitor (NNI)pocket of reverse transcriptase (RT)” means a compound thatsubstantially enters and binds the NNI binding site on RT. In oneembodiment, a compound that fits the NNI pocket of RT inhibits RTactivity. Generally, compounds which better fit the NNI pocket of RTcontact a greater portion of the available molecular surface of thepocket and are more potent inhibitors of RT activity. In one embodiment,the compound that fits the NNI pocket of RT is a PETT analog. In anotherembodiment, the compound that fits the NNI pocket of RT is a DABOanalog. In another embodiment, the compound that fits the NNI pocket ofRT is a HEPT analog.

As used herein, “docking” a compound in a binding pocket meanspositioning a model of a compound in a model of the binding pocket. Inone embodiment, the model of the binding pocket can be a compositebinding pocket constructed in accordance with the invention. The modelof the binding pocket can be, for example, based on coordinates obtainedfrom the crystal structure of RT complexed with a NNI. In oneembodiment, the docking is performed with the use of computer software,such as the Affinity program within InsightII (Molecular SimulationsInc., 1996, San Diego, Calif.). Docking permits the identification ofpositions of the compound within the binding pocket that are favored,for example, due to minimization of energy.

As used herein, “minimization of energy” means achieving an atomicgeometry of a molecule or molecular complex via systematic alterationsuch that any further minor perturbation of the atomic geometry wouldcause the total energy of the system as measured by a molecularmechanics force-field to increase. Minimization and molecular mechanicsforce-fields are well understood in computational chemistry (Burkert, U.and Allinger, N. L., Molecular Mechanics, ACS Monograph, 1982, 177,59-78, American Chemical Society, Washington, D.C.).

As used herein, “comparing” includes visualizing or calculatingavailable space encompassed by the molecular surface of the compositebinding pocket of the invention, taking into account the flexibility ofresidues, such as Tyr180, Tyr181, Tyr318, Tyr319, Phe227, Leu234,Trp229, Pro95, and Glu138 of RT (the latter from the p51 subunit of RT).“Comparing” also includes calculating minimal energy conformations.

As used herein, “gap space” means unoccupied space between the van derWaals surface of a compound positioned within the binding pocket and thesurface of the binding pocket defined by residues in the binding site.This gap space between atoms represents volume that could be occupied bynew functional groups on a modified version of the compound positionedwithin the binding pocket.

In the present invention, the terms “analog” or “derivative” are usedinterchangeably to mean a chemical substance that is relatedstructurally and functionally to another substance. An analog orderivative contains a modified structure from the other substance, andmaintains the function of the other substance, in this instance,maintaining the ability to interact with an NNI-RT binding site. Theanalog or derivative need not, but can be synthesized from the othersubstance. For example, a HEPT analog means a compound structurallyrelated to HEPT, but not necessarily made from HEPT.

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms.

As used herein, “alkene” includes both branched and straight-chainunsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms.

As used herein, “halogen” includes fluoro, chloro, bromo and iodo.

As used herein “non-hydrogen atom group” includes, but is not limitedto, alkyl, alkenyl, alkynyl, halo, hydroxy, alkoxy, thiol, thiolalkyl,amino, substituted amino, phosphino, substituted phosphino, or nitro. Inaddition, cycloalkyl, aryl, and aralkyl groups may be included if thenon-hydrogen atom group contains a sufficient number of non-hydrogenatoms. Often, a number or range of numbers is specified to indicate thenumber of non-hydrogen (e.g., C, O, N, S, or P) atoms in the functionalgroup.

As used herein, “pharmaceutically acceptable salt” refers to a salt thatretains the desired biological activity of the parent compound and doesnot impart any undesired toxicological effects. Examples of such saltsinclude, but are not limited to, (a) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; and saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, furmaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids,naphthalenedisulfonic acids, polygalacturonic acid; (b) salts withpolyvalent metal cations such as zinc, calcium, bismuth, barium,magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or(c) salts formed with an organic cation formed fromN,N′-dibenzylethylenediamine or ethylenediamine; or (d) combinations of(a) and (b) or (c), e.g., a zinc tannate salt; and the like. Thepreferred acid addition salts are the trifluoroacetate salt and theacetate salt.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with a compound of the invention, allowsthe compound to retain biological activity, such as the ability toinhibit RT activity, and is non-reactive with the subject's immunesystem. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsion, and various types ofwetting agents. Preferred diluents for aerosol or parenteraladministration are phosphate buffered saline or normal (0.9%) saline.

Compositions comprising such carriers are formulated by well knownconventional methods (see, for example, Remington's PharmaceuticalSciences, Chapter 43, 14th Ed., Mack Publishing Col, Easton Pa. 18042,USA).

Composite Binding Pocket of the Invention

As shown in FIG. 1, the NNI binding site of HIV-RT rests between thepalm and thumb regions of the RT molecular structure, adjacent to thehinge region. The NNI binding site includes two distinct regions,indicated in FIG. 1B as Wing 1 and Wing 2, forming a butterfly-shapedbinding pocket.

In the method of the invention, a composite ligand binding pocket isconstructed by superimposing multiple structures of ligand-binding sitecomplexes, preferably using 5 or more distinct structures. In oneembodiment, the composite ligand binding pocket is an HIV-1reverse-transcriptase (RT) nonnucleoside inhibitor (NNI) binding pocketconstructed by superimposing multiple structures of NNI-RT complexes.The composite binding pocket is preferably an HIV-1 RT-NNI bindingpocket.

A preferred binding pocket of the invention can be made bysuperimposition of coordinates, obtainable from the Protein Data Bank(PDB) via access codes disclosed herein, corresponding to thethree-dimensional structure of an RT-NNI complex. The superimposition ofcoordinates is preferably based on alignment of the coordinatescorresponding to the palm region of the binding pocket due to thegreater rigidity of this region.

The superimposing of coordinates can also be accomplished by first usingmodels of the protein backbone and DNA phosphate backbone of the RT-DNAcomplex structure (with PDB access code hmi) onto a model of an RTmutant complexed with an NNI, such as APA((2-acetyl-5-methylanilino)(2,6-dibromophyl)acetamide) having PDB accesscode hni. Next, models of one or more additional RT-NNI complexes aresuperimposed onto the models superimposed above. In one embodiment, thesuperimposition is based on alignment of the region of RT from residue100 to 230, preferably by a least squares procedure. In anotherembodiment, the superimposition is based on alignment of the region ofRT from residues 97 to 213. Preferably, the superimposition is based onalignment of the palm region and part of the NNI binding site. Mostpreferably, the superimposition is based on alignment of the regioncorresponding to residues 100 to 230 of RT, or on alignment of 117 Calpha atoms of residues 97 to 213, and preferably using a least squaresprocedure.

A molecular surface of a binding pocket can then be generated thatencompasses all superimposed NNI models. One such composite bindingpocket constructed from nine individual NNI-RT complex structures, isshown in FIG. 2A. Grid lines in the figure represent the collective vander Waals surface, and highlight space available for binding.

The molecular surface of the complex can be generated, for example, byreading the overlaid coordinates of the complexed inhibitors into acomputer program such as GRASP (A. Nicholls, GRASP, GraphicalRepresentation and Analysis of Surface Properties, 1992, New York).Examples of NNI compounds which can be used in the construction of abinding pocket include, but are not limited to, HEPT, MKC, TNK, APA,Nevirapine, N-ethyl Nevirapine derivative, 8-Cl TIBO, and 9-Cl TIBO (PDBaccess codes, rti, rt1, rt2, hni, vrt, rth, hnv and rev or tvr,respectively).

Using the composite NNI binding pocket, binding of compounds can bemodeled to identify available space within the binding pocket. New andmore potent NNI inhibitors of RT can be developed by designing compoundsto better fit the binding pocket.

In one embodiment, the composite binding pocket is constructed bysuperimposing structures of NNI-RT complexes comprising RT complexedwith: an HEPT or MKC analog; a TNK analog; an APA analog; a Nevirapineanalog; and a TIBO analog. In another embodiment, the composite NNIbinding pocket is based on the structure of RT complexed with 9 NNI andon the RT-DNA complex. Examples of NNI compounds which can be used inthe construction of a binding pocket include, but are not limited to,HEPT, MKC, TNK, APA, Nevirapine, N-ethyl Nevirapine derivative, 8-ClTIBO, and 9-Cl TIBO structures (PDB access codes, rti, rt1, rt2, hni,vrt, rth, hnv, and tvr and/or rev, respectively). In one embodiment, theresulting composite binding pocket has the coordinates set forth inTable 9.

Construction and Use of the Binding Pocket

A compound that binds the NNI binding site of reverse transcriptase isidentified by comparing a test compound to the composite binding pocketof the invention and determining if the compound fits the bindingpocket. As shown in FIGS. 7A and 7B, the test compound may be comparedto another inhibitory compound, by superimposing the structures in thebinding pocket. The test compound is also compared to the binding pocketby calculating the molecular surface of the compound complexed with thecomposite binding pocket. The extent of contact between the molecularsurface of the compound and the molecular surface of the binding pocketcan be visualized, and the gap space between the compound and thebinding pocket can be calculated. In FIGS. 4A and 4B, gaps between themolecular surface of the binding pocket and the NNI are presented inred, with each red line being 1 angstrom in distance.

To design a novel inhibitor of reverse transcriptase, a compound isdocked in the composite binding pocket of the invention. Gap space isidentified between the compound and the binding pocket, for example,using an algorithm based on a series of cubic grids surrounding thedocked compound, with a user-defined grid spacing. The compound is thenmodified to more completely occupy the gap space.

Computerized docking procedures can be used to dock the test compound inthe binding pocket and analyze the fit. One docking program, DOCK(Kuntz, I. D., et al., J. Mol. Biol., 1982, 161, 269-288; available fromUniversity of California, San Francisco), is based on a description ofthe negative image of a space-filling representation of the receptorthat should be filled by the ligand. DOCK includes a force-field forenergy evaluation, limited conformational flexibility and considerationof hydrophobicity in the energy evaluation. CAVEAT (Bartlett, P. A. etal., Molecular Recognition in Chemical and Biological Problems, SpecialPub., Royal Chem. Soc., 1989, 78, 182-196; available from University ofCalifornia, Berkeley) suggests ligands to a particular receptor based ondesired bond vectors. HOOK (Molecular Simulations, Burlington, Mass.)proposes docking sites by using multiple copies of functional groups insimultaneous searches. MACCS-3D (Martin, Y. C., J. Med. Chem., 1992, 35,2145-2154) is a 3D database system available from MDL InformationSystems, San Leandro, Calif. Modeling or docking may be followed byenergy minimization with standard molecular mechanics forcefields ordynamics with programs such as CHARMM (Brooks, B. R. et al., J. Comp.Chem., 1983, 4, 187-217) or AMBER (Weiner, S. J. et al., J. Am. Chem.Soc., 1984, 106, 765-784).

LUDI (Bohm, H. J., J. Comp. Aid. Molec. Design, 1992, 6, 61-78;available from Biosym Technologies, San Diego, Calif.) is a programbased on fragments rather than on descriptors. LUDI proposes somewhatlarger fragments to match with the interaction sites of a macromoleculeand scores its hits based on geometric criteria taken from the CambridgeStructural Database (CSD), the Protein Data Bank (PDB) and on criteriabased on binding data. Other software which can be used to proposemodifications for constructing novel inhibitors include LEGEND(Nishibata, Y. and Itai, A., Tetrahedron, 1991, 47, 8985; available fromMolecular Simulations, Burlington, Mass.) and LeapFrog (TriposAssociates, St. Louis, Mo.).

The AUTODOCK program (Goodsell, D. S. and Olson, A. J., Proteins:Struct. Funct. Genet., 1990, 8, 195-202; available from Scripps ResearchInstitute, La Jolla, Calif.) helps in docking ligands to their receptiveproteins in a flexible manner using a Monte Carlo simulated annealingapproach. The procedure enables a search without bias introduced by theresearcher. This bias can influence orientation and conformation of aligand in the active site. The starting conformation in a rigid dockingis normally biased towards an energy minimum conformation of the ligand.However, the binding conformation of the ligand may be of relativelyhigh conformational energy, but offset by the binding energy.

In a preferred embodiment of the invention, docking is performed byusing the Affinity program within InsightII (Molecular Simulations Inc.,1996, San Diego, Calif.). As modeling calculations progress during thedocking procedure, residues within a defined radius of 5 Å from the NNImolecule are allowed to move in accordance with energy minimization,permitting the identification of promising positions for modification.Initial coordinates of newly designed compounds can be generated usingthe Sketcher module within InsightII.

In one embodiment, the method further comprises calculating theinhibition constant of the docked compound. Inhibition constants (K_(i)values) of compounds in the final docking positions can be evaluatedusing a score function in the program, LUDI (Bohm, H. J., J. Comput.Aided Mol. Des., 1994, 8, 243-256; Bohm, H. J., J. Comput. Aided Mol.Des., 1992, 6, 593-606). Predictions of K_(i) values can be improved bymodifications of the LUDI calculation., for example, those described inExample 1. First, the molecular surface area can be directly calculatedfrom the coordinates of the compounds in docked conformation using theMS program described in Connolly, M. L., 1983 Science 221: 709-713.Second, because InsightII does not account for structural rigidityimposed by internal hydrogen bonds, the number of rotatable bonds can bere-evaluated. For example, this re-evaluation can be performed bycounting the number of rotatable bonds according to the principleintroduced by Bohm (supra) and taking out the number of bonds which arenot rotatable due to the conformational restraint imposed by theinternal hydrogen bond between the thiourea NH and pyridyl N in PETTderivatives. Third, the calculation can be modified by the assumptionthat the conserved hydrogen bond with RT does not deviate significantlyfrom the ideal geometry. This assumption is supported by the fact that,in known crystal structures of RT complexes, all hydrogen bonds betweenNNIs and RT are near the ideal geometry. These constraints provide formore predictive K_(i) values for modeled compounds.

In a preferred embodiment, the compound has a predicted inhibitionconstant (K_(i)) of less than about 1 μM, and the compound in thebinding has an estimated molecular surface area greater than 276 Å².

Candidate inhibitors of RT identified or designed by the methods of theinvention can be evaluated for their inhibitory activity usingconventional techniques which typically involve determining the locationand binding proximity of a given moiety, the occupied space of a boundinhibitor, the deformation energy of binding of a given compound andelectrostatic interaction energies. Examples of conventional techniquesuseful in the above evaluations include, but are not limited to, quantummechanics, molecular dynamics, Monte Carlo sampling, systematic searchesand distance geometry methods (Marshall, G. R., Ann. Ref Pharmacol.Toxicol., 1987, 27, 193). Examples of computer programs for such usesinclude, but are not limited to, Gaussian 92, revision E2 (Gaussian,Inc. Pittsburgh, Pa.), AMBER version 4.0 (University of California, SanFrancisco), QUANTA/CHARMM (Molecular Simulations, Inc., Burlington,Mass.), and Insight II/Discover (Biosym Technologies Inc., San Diego,Calif.). These programs may be implemented, for example, using a SiliconGraphics Indigo2 workstation or IBM RISC/6000 workstation model 550.Other hardware systems and software packages will be known and ofevident applicability to those skilled in the art.

Inhibitors identified or designed by the methods of the invention can betested for their anti-HIV or anti-RT activity using one of the standardin vitro assays known in the art, such as the p24 enzyme immunoassaydisclosed herein.

The invention further provides novel compounds identified by the abovemethods, which can be used as inhibitors of RT. Novel inhibitors soidentified include analogs or derivatives of known NNI compounds such asHEPT, DABO, and PETT, as well as novel compounds designed to fit thecomposite binding pocket which are unrelated to any known NNI compound.

Compounds of the Invention

Compounds of the invention are useful as nonnucleoside inhibitors of RT.These include, for example, analogs and derivatives of PETT, DABO, andHEPT compounds, as well as novel compounds unrelated to known NNI butdesigned to fit the composite binding pocket.

PETT Compounds:

Novel compounds of the invention include derivatives and analogs ofPETT, having the general formula (I):

Z can be phenyl, piperizine, piperidine, or morpholine. Z is preferablysubstituted with one or more substituents, including alkyl, alkene,halogen, methoxy, alcohol, amino, thio, thioxy, or phosphino. In oneembodiment, the compounds of the invention are PETT derivatives oranalogs having the following formula (II):

The R's can be the same or different, and represent points of optionalsubstitution. R₂, R₃, R₄, R₅, R₆, R₇ and R₈ can be hydrogen, or can besubstituted, with a non-hydrogen atom group such as halo (Br, Cl, F, I),alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, orRNH₂ group, where R is alkyl. Preferably, one or more is alkyl, halo, oralkoxy. Preferred halogens are F, Br, or Cl. One or more of R₂, R₃, R₄,R₅, R₆, and R₇ can be a C1-C3 alkoxy, e.g., methoxy.

R₈ can also be aryl, aralkyl, ROH, or RNH₂ group, where R is alkyl.Preferably, at least one of R₂, R₃, R₄, R₅, and R₆ is not hydrogen. R₄is a preferably hydrophobic group such as H, an alkyl or alkene, and canbe Me, Et, or i-Pr. R₆ and/or R₇ are preferably a 3 or 4(non-hydrogen)-atom group.

R₆ and R₇ can be a group having 1 to 4 non-hydrogen atoms, whereas R₂,R₃, and R₅ preferably each are a group having 1 to 3 non-hydrogen atoms.Available gap space in the binding pocket near R₈, is approximately 8angstroms by 5 angstroms, by 3.3 angstroms. Thus, a molecule having avolume of up to about 8×6×4 angstroms can be used to fill this space,e.g., accommodating a group of about 7 non-hydrogen atoms, or up toabout the size of a phenyl ring. R₈ can be halo, alkyl, phenyl, —CH₂Ph,or alkoxy. R₈ can be X—R, where X is a bridging atom, including, but notlimited to, C, S, O, N and P.

In a preferred embodiment, R₈ is bromine, and at least one of R₂, R₃,R₄, R₅, and R₆ is fluoro, chloro, or methoxy.

A compound of the invention preferably conforms to the composite NNIbinding pocket of the invention. Most preferably, the compound complexedwith an NNI-RT binding pocket, has a predicted K_(i) of less than about1 μM.

Preferred modifications of PETT compounds include ortho-halogen,meta-O-Me, and hydrophobic groups at the para position of the ring. Mostpreferably, the modifications do not disrupt the intramolecular hydrogenbond. Specific compounds include those having the following formulae(III-VIII) shown below.

In another embodiment, the PETT derivative comprises the formula (IX):

The R's can be the same or different, and represent points of optionalsubstitution. R₅, R₆, and R₇ can be hydrogen, or can be substituted,with a non-hydrogen atom group such as halo (Br, Cl, F, I), alkyl,alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH₂group, where R is alkyl. Preferably, one or more is alkyl, halo, oralkoxy. Preferred halogens are F, Br, or Cl. One or more of R₅R₆, and R₇can be a C1-C3 alkoxy, e.g., methoxy.

R₆ and/or R₇ are preferably a 3 or 4 (non-hydrogen)-atom group. R₆ andR₇ can be a group having 1 to 4 non-hydrogen atoms, whereas R₅preferably is a group having 1 to 3 non-hydrogen atoms. R₈ can be agroup of about 7 non-hydrogen atoms, or up to about the size of a phenylring. R₈ can be hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy,thioalkyl, thiol, phosphino, aryl, aralkyl, —CH₂Ph, alkoxy, ROH or RNH₂,where R is alkyl. R₈ can be X—R, where X is a bridging atom, including,but not limited to, C, S, O, N and P.

X can be CR′R″, NR′″, or O, where R′, R″, and R′″ can be hydrogen, halo,alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, or phosphino group.In one embodiment, R₅, R₆, R′, R″, and R′″ are each hydrogen. In analternative embodiment, X is CR′R″ and at least one of R′ and R″ arefluoro, chloro, bromo, hydroxy, methoxy, or C1-3 alkyl. In a preferredembodiment, R₈ is bromine, and at least one of R₅, R₆, and R₇ is fluoro,chloro, or methoxy.

Preferred compounds include a larger functional group near the ethyllinker, for example R₇ acetamide or methoxy. Also preferred is a bulkierheterocyclic ring such as a bulky piperidinyl ring or an ortho/metasubstituted pyridyl ring.

Specific PETT derivatives of the invention include:

-   N-[2-(1-piperidinoethyl)]-N′-[2-(5-bromopyridyl)]thiourea,-   N-[2-(2,5-dimethoxyphenethyl)]-N′-[2-(5-bromopyridyl)]thiourea,-   N-[2-(o-Chlorophenethyl)]-N′-[2-(5-bromopyridyl)]thiourea    N-[2-(o-Fluorophenethyl)]-N′-[2-(5-bromopyridyl)]thiourea, and-   N-[2-(m-Fluorophenethyl)]-N′-[2-(5-bromopyridyl)]thiourea.

Other specific compounds of the invention are described in the Examplesbelow.

DABO Compounds:

In another embodiment of the invention, the compounds are derivatives ofDABO, and have the following general formula (X):

R₁ and R₂ can be alike or different, and can be hydrogen, halo, alkyl,alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNHgroup, where R is alkyl. Preferably, one or more of R₁ and R₂ is a C1-3alky, such as methyl (Me), ethyl (Et), or isopropyl (i-Pr). Preferably,R₁ is alkyl, alkenyl, ROH, or RNH₂. R₂ is preferably halo, alkyl, orC1-3 alkoxy.

Y can be S or O, and is preferably S. R3 can be alkyl, alkenyl, aryl,aralkyl, ROH, or RNH group, where R is alkyl, and is preferably C1-3alkyl.

Specific DABO compounds of the invention include:

-   5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one.    Other specific compounds of the invention are described in the    Examples below.    HEPT Compounds:

In another embodiment, the compounds of the invention are HEPTderivatives having the formula (XI):

X and Y can be independently S or O. Preferably, at least one of X and Yis S. More preferably, X is S, and in specific embodiments, both X and Yare S.

R₁ and R₂ can be hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy,thioalkyl, thiol, phosphino, ROH, or RNH group, where R is alkyl. R₃ canbe H, alkyl, alkenyl, aryl, aralkyl, ROH, or RNH group, where R isalkyl. Preferably, R₁ is alkyl, alkenyl, ROH, or RNH₂, and can be, forexample, methyl, ethyl, or isopropyl. R₂ is preferably halo, alkyl, orC1-3 alkoxy, and is preferably in the ortho or meta position. R₂ can beBr, F, Cl, or O-Me.

Specific HEPT compounds of the invention include:

-   6-benzyl-5-isopropyl-1[(methylthio)methyl]-2-thiouracil.

Other specific compounds of the invention are described in the Examplesbelow.

The compounds of the invention have the ability to inhibit replicationof a retrovirus, such as human immunodeficiency virus (HIV), preferablywith an IC₅₀ of less than 50 μM, for example, as determined by p24enzyme immunoassay described in the Examples below. More preferably, thecompound of the invention inhibits replication of HIV in the p24 assaywith an IC₅₀ of 1 to 5 μM, or less. Most preferably, the compoundinhibits replication of HIV in the p24 assay with an IC₅₀ of less than 5nM. In some embodiments, the compound inhibits replication of HIV in thep24 assay with an IC₅₀ of less than 1 nM.

The invention provides a composition comprising a compound or inhibitorof the invention, and optionally, an acceptable carrier. The compositioncan be a pharmaceutical composition. Compositions of the invention areuseful for prevention and treatment of retroviral infection, such as HIVinfection.

Methods of Using Compounds of the Invention

The compounds of the invention are useful in methods for inhibitingreverse transcriptase activity of a retrovirus. Retroviral reversetranscriptase is inhibited by contacting RT in vitro or in vivo, with aneffective inhibitory amount of a compound of the invention. Thecompounds of the invention also inhibit replication of retrovirus,particularly of HIV, such as HIV-1. Viral replication is inhibited, forexample, by contacting the virus with an effective inhibitory amount ofa compound of the invention.

Due to the ability to inhibit replication of retrovirus and to inhibitretroviral RT activity, the invention provides a method for treating orpreventing retroviral infection, such as HIV infection, and a method fortreating AIDS or AIDS-related complex (ARC). The method comprisesadministering to a subject an effective inhibitory amount of a compoundof the invention or a pharmaceutically acceptable salt of the compound.The compound or inhibitor of the invention is preferably administered incombination with a pharmaceutically acceptable carrier, and may becombined with specific delivery agents, including targeting antibodiesand/or cytokines. The compound or inhibitor of the invention may beadministered in combination with other antiviral agents,immunomodulators, antibiotics or vaccines.

The compounds of the invention can be administered orally, parentally(including subcutaneous injection, intravenous, intramuscular,intrasternal or infusion techniques), by inhalation spray, topically, byabsorption through a mucous membrane, or rectally, in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants or vehicles. Pharmaceutical compositionsof the invention can be in the form of suspensions or tablets suitablefor oral administration, nasal sprays, creams, sterile injectablepreparations, such as sterile injectable aqueous or oleagenoussuspensions or suppositories.

For oral administration as a suspension, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can contain microcrystalline cellulose forimparting bulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners or flavoringagents. As immediate release tablets, the compositions can containmicrocrystalline cellulose, starch, magnesium stearate and lactose orother excipients, binders, extenders, disintegrants, diluents andlubricants known in the art.

For administration by inhalation or aerosol, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can be prepared as solutions in saline,using benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons or othersolubilizing or dispersing agents known in the art.

For administration as injectable solutions or suspensions, thecompositions can be formulated according to techniques well-known in theart, using suitable dispersing or wetting and suspending agents, such assterile oils, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

For rectal administration as suppositories, the compositions can beprepared by mixing with a suitable non-irritating excipient, such ascocoa butter, synthetic glyceride esters or polyethylene glycols, whichare solid at ambient temperatures, but liquify or dissolve in the rectalcavity to release the drug.

Dosage levels of approximately 0.02 to approximately 10.0 grams of acompound of the invention per day are useful in the treatment orprevention of retroviral infection, such as HIV infection, AIDS or ARC,with oral doses 2 to 5 times higher. For example, HIV infection can betreated by administration of from about 0.1 to about 100 milligrams ofcompound per kilogram of body weight from one to four times per day. Inone embodiment, dosages of about 100 to about 400 milligrams of compoundare administered orally every six hours to a subject. The specificdosage level and frequency for any particular subject will be varied andwill depend upon a variety of factors, including the activity of thespecific compound the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, and diet of thesubject, mode of administration, rate of excretion, drug combination,and severity of the particular condition.

The compound of the invention can be administered in combination withother agents useful in the treatment of HIV infection, AIDS or ARC. Forexample, the compound of the invention can be administered incombination with effective amounts of an antiviral, immunomodulator,anti-infective, or vaccine. The compound of the invention can beadministered prior to, during, or after a period of actual or potentialexposure to retrovirus, such as HIV.

Strategies for Design and Synthesis of Inhibitors

It has been proposed that NNI interfere with reverse transcription byaltering either the conformation or mobility of RT rather than directlypreventing the template-primer binding (Tantillo, C. et al., J Mol Biol,1994, 243, 369-387). Specifically, binding of NM to the NNI binding site(approximately 10 Å away from the polymerase catalytic site) inhibits RTby interfering with the mobility of the “thumb” and/or position of the“primer grip” (residues 229-231), which interact with the DNA primerstrand (FIG. 1A).

Computer programs can be used to identify unoccupied (aqueous) spacebetween the van der Waals surface of a compound and the surface definedby residues in the binding site. These gaps in atom-atom contactrepresent volume that could be occupied by new functional groups on amodified version of the lead compound. More efficient use of theunoccupied space in the binding site could lead to a stronger bindingcompound 1f the overall energy of such a change is favorable. A regionof the binding pocket which has unoccupied volume large enough toaccommodate the volume of a group equal to or larger than a covalentlybonded carbon atom can be identified as a promising position forfunctional group substitution. Functional group substitution at thisregion can constitute substituting something other than a carbon atom,such as oxygen. If the volume is large enough to accommodate a grouplarger than a carbon atom, a different functional group which would havea high likelihood of interacting with protein residues in this regionmay be chosen. Features which contribute to interaction with proteinresidues and identification of promising substitutions includehydrophobicity, size, rigidity and polarity. The combination of docking,K_(i) estimation, and visual representation of sterically allowed roomfor improvement permits prediction of potent derivatives.Design of HEPT Derivatives

New HEPT derivative designs included compounds with added groups at theN-1 (Y—R3) and C-5 (R₁) positions and those having oxygen (X or Y) atomsreplaced by sulfur. Substitution of oxygen by sulfur can aid binding bydecreasing the desolvation energy involved in binding. The modificationswere made such that the HEPT derivative would fit favorably into thebutterfly-shaped RT-NNI binding site, (See FIG. 2A) with the benzyl ringresiding in one wing and thymine ring in the other. For all designedcompounds, the benzyl ring is near Trp229 and the N−1 group is nearPro236, a typical position observed in crystal structures. The modelingcalculations, along with the application of the constructed bindingpocket, provided a guideline for the synthesis of lead compoundsdesigned to have potent anti-HIV activity. The choice of compounds wasalso based on synthetic feasibility.

The region of the NNI site of HIV-1 RT located near the thymine ringnitrogen N−1 of the HEPT analogs contains a Pro236 loop region which islarge enough to accommodate N−1 substituents. When an inhibitor binds tothe NNI site of HIV-I RT, the presence of a hydrophobic N−1 substituentcould influence the Pro loop of this flexible region and provideadditional hydrophobic contact leading to stronger binding. Dockingresults indicated that substitution at N−1 also helps the moleculeposition itself to achieve the best fit within the pocket.

The LUDI analysis showed a substantial increase in contact (lipo score)between the compound and the pocket and the calculation suggested anincrease in hydrophobic contact and stronger binding when thesubstituent on the N−1 tail (R₃) is larger in size than a methyl moiety.

The Tyr183 residue of the HIV-1 RT is located in the catalytic regionwhich has a conserved YMDD motif characteristic of reversetranscriptases. Therefore, the displacement of this tyrosine residue caninterfere with catalysis and render the HIV-1 RT protein inactive. Ithas been suggested that bulky substituents at the 5th position of thethymine ring (R₁) could indirectly accomplish this goal by displacingTyr181 which is near Tyr183. The composite binding pocket showssufficient room for at least a 3-carbon group in this region. Theaddition of a methyl, ethyl or isopropyl group on the 5th position ofthe thymine ring would lead to a higher affinity for the relativelyhydrophobic environment.

LUDI analysis showed that the hydrophobic contact increases ashydrophobic groups at the 5th position (R₁) get bulkier. As it binds tothe site, the ethyl or isopropyl group causes the nearby Tyr181 residueto rotate away from the inhibitor. This change in conformation in turnaffects the positions of the neighboring Tyr183 and Tyr188 which canlead to the inactivation of HIV-1 RT.

DABO Derivatives

Detailed analysis of HEPT binding revealed that the N1 substituents ofHEPT derivatives occupy the same region of the binding site as the thio(S2) substituents of DABO compounds (See FIG. 7A). Therefore, new DABOderivatives were designed and their binding into the NNI site of RTmodeled using the crystal structure coordinates of the RT/MKC complex(pdb access code: rt1) and a molecular docking procedure. The finalcoordinates of the docked molecules were then superimposed into thecomposite binding pocket to evaluate the fit within the RT NNI pocket.Notably, multiple sterically allowed unoccupied spatial gaps in thebinding site were identified from the docking studies which could befilled by strategically designed functional groups (See FIG. 7B).

The docked DABO molecule showed significant space surrounding the6-benzyl ring and the 5th position of the thymine ring, which led to ourdesign and synthesis of new DABO derivatives. Specific DABO compoundsare discussed more fully in the Examples, below.PETT Derivatives

Each PETT derivative described in the Examples below, can be viewed astwo chemical groups linked together by a thiourea group. Upon bindingRT, the PETT derivative fits into the butterfly-shaped binding site.(See FIG. 6). One half of the molecule is composed of a pyridyl thioureagroup (compounds I-1 to 4, II-1 to 9, and III-1 to 3) or a2-aminothiazole group (PETT) which forms an intramolecularhydrogen-bonded 6-membered heterocyclic ring (shown below). The otherhalf of the molecule is a piperidinyl ring (II-9), a pyridyl ring(trovirdine), or a phenyl ring separated from the thiocarbonyl group byan ethyl linker.

The positions of the compounds having stronger binding and higher scores(evaluated by LUDI function) all fall into the butterfly-shaped bindingregion with one part residing in Wing 1 and the other in Wing 2, asillustrated in FIG. 1B. For these compounds the ring closest to thethiocarbonyl group is near the Lys(K) 101 loop and the other pyridylring is near Trp(W)229 derivatives.

Analysis of trovirdine, revealed multiple sites which can be used forthe incorporation of larger functional groups. In the composite bindingpocket, the docked trovirdine molecule showed a lot of usable spacesurrounding the pyridyl ring, (R₂-R₆), the ethyl linker (R₇) and nearthe 5-bromo position (R₈). (See FIG. 5A).

Efficient use of this space by strategically designed functional groupswould lead to high affinity binding and ultimately result in betterinhibitors. Our modeling studies suggest that designs using the spaceavailable in these regions, including (1) substitutions at R₂-R₆; (2)substituting heterocyclic rings for the pyridyl ring of trovirdine; (3)substitutions at R₇; (4) substitutions at R₈; and (5) maintaining theintramolecular hydrogen bond. As shown in the Examples below,modifications in these areas lead to potent RT inhibitors.

Advantages of the Invention

The invention provides a model for the three-dimensional structure ofthe RT-DNA complex based on the available backbone structure of RT-DNAcomplex and full structure of RT complexed with several NNI compounds.This is the first model to combine structural information from severalcomplexes into a single composite and provides a suitable working modelfor the development of novel inhibitory compounds. The use of multipleNNI binding site coordinates from RT-NNI structures, as disclosedherein, permits the generation of a composite molecular surface.Analysis of the composite NNI binding pocket of the invention revealsthat the binding pocket is surprisingly and counter-intuitively larger(instead of smaller) and more flexible (instead of more rigid) thanexpected. This composite NNI binding pocket serves as a guide for thesynthesis and analyses of structure-activity relationships for theidentification and design of new and more potent NNI of RT. Thecomposite binding pocket additionally provides a model for the design ofderivatives of NNIs for which crystal structure information is notavailable (e.g., PETT, DABO).

The compounds of the invention are useful for inhibition of RT activityand for inhibition of retroviral replication. The compounds disclosedherein provide more potent NNI of RT than known HEPT, DABO and PETTderivatives. With all strategies combined, a number of sites areidentified for developing more potent derivatives of PETT, such as theincorporation of a larger functional group near the ethyl linker ofPETT. Hitherto unknown piperidinyl substituted and piperozinylsubstituted, as well as morpholinyl substituted PETT derivatives aredisclosed which show potent anti-HIV activity at nanomolarconcentrations.

In addition, the compounds of the invention provide a higher selectivityindex (S.I.>10⁵) than currently available anti-HIV compounds. This highS.I. permits more effective antiviral activity with a minimum of adversecytotoxic effects.

EXAMPLES

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Example 1 Modeling Procedure

Construction of the Composite NNI Binding Pocket

A novel model of the NNI binding pocket of RT was constructed bysuperimposing nine individual RT-NNI crystal structures and thengenerating a van der Waals surface which encompassed all of the overlaidligands. This “composite binding pocket” surprisingly reveals adifferent and unexpectedly larger NNI binding site than shown in orpredictable from any of the individual structures and serves as a probeto more accurately define the potentially usable space in the bindingsite (FIG. 2A).

Modeling studies were based on the construction of a binding pocketwhich encompassed the superimposed crystal structure coordinates of allknown RT-NNI complexes, including nine different structures of RTcomplexed with HEPT, MKC, TNK, APA, Nevirapine, N-ethyl Nevirapinederivative, 9-Cl TIBO (Ren, J. et al., Structure, 1995, 3, 915-926);9-Cl TIBO (Das, K. et al., J. Mol. Biol., 1996, 264, 1085-1100) and8-Cl-TIBO (PDB access codes rti, rt1, rt2, hni, vrt, rth, rev, tvr, andhnv, respectively).

The “thumb” region of RT complexes are relatively variable compared withthe “palm” region. Therefore, a total of 117 C-alpha atoms of theresidues from 97 to 213 which cover part of the NNI binding site and the“palm” region were used for a least-squares superimposing procedurewithin the program O (Jones, T. A. et al., Acta Crystallogr. A., 1991,47, 110-119). Each coordinate set was superimposed onto the same initialcoordinate set (RT/9-Cl TIBO). the distance between the pair wasminimized by rotating and translating one coordinate set onto the other,minimizing distances between x, y, and z coordinates, according to themethod of the program “O”. The root mean square (RMS) values of thecoordinates of the atoms being superimposed are shown to be 1.00, 0.98,0.99, 0.62, 0.80, 0.87, 0.94 and 0.65 Å for HEPT, MKC, TNK, APA,Cyclopropanyl Nevirapine, N-ethyl Nevirapine derivative and two 9-ClTIBO compounds, respectively. Next, the coordinates of the correspondinginhibitor molecules were then transformed according to the same matricesderived from the superimposition. Lastly, the overlaid coordinates ofall inhibitors were read into the program GRASP (Nicholls, A., GRASP1992, New York), from which an overall molecular surface was generatedproviding a binding pocket encompassing all inhibitors.

As shown in FIG. 2A, the surface of the binding pocket was color codedto reflect characteristics of the overlaid inhibitors, such as hydrogenbonding, hydrophilic, and hydrophobic regions. The amide nitrogens onthe uracil ring of HEPT and TIBO derivatives are color-coded red forhydrogen bonding atoms. Oxygen or sulfur atoms of carbonyl,thiocarbonyl, and ester groups, nitrogen atoms of amine groups, andhalogen atoms are color-coded blue for polar (hydrophilic) groups.Carbon atoms are considered hydrophobic and are colored grey. Thispocket, referred to as the composite binding pocket, was used as a basisfor the analysis of inhibitor binding.

To generate the coordinates of the composite binding pocket using theInsightII program, each data point of the net defining the surface ofthe, pocket was represented as a water molecule and was saved inBrookhaven Protein Databank (pdb) format. To provide a visual frame ofreference, the coordinates have been superimposed on the pdb coordinatesof an existing crystal structure having pdb access code hnv (HIV-1RT/8-Cl TIBO complex). The coordinates of a composite binding pocket forHIV-1 RT generated by superimposing nine different NNI-RT complexes, areset forth in Table 9.

Docking and K_(i) Prediction

A computer simulation of the binding of PETT, DABO, and HEPT compoundsinto the NNI binding site of RT was accomplished using a moleculardocking procedure. Docking of the compounds into the NNI binding siterequired the use of X-ray coordinates of an RT-NNI complex (RT/9-Cl-TIBOcomplex was used for modeling PETT, and the RT/MKC-442 complex was usedfor modeling DABO and HEPT). Upon binding to RT, the compound can fitinto a butterfly-shaped NNI binding site (described by Ding et. al),Ding, J. et al., Nat. Struct. Biol., 1995, 2, 407-415 (FIGS. 1B and 2A).Once the final docked position of the molecule in the NNI site wasdetermined, the molecule was assigned a score (LUDI), from which anestimation of the inhibition constant (K_(i) value) was determined.

After docking and K_(i) estimation was completed for the inhibitors,evaluation of the docked compounds in the active site of RT involvedplacing each compound into the composite binding pocket using the sameorientation matrix utilized in construction of the pocket. Thepotentially flexible regions in the binding site were then readilyidentified as were atom sites for future derivatization of thecompounds. Fixed docking in the Affinity program within InsightII(InsightII, Molecular Simulations Inc., 1996, San Diego, Calif.), wasused for docking small molecules to the NNI binding site which was takenfrom a crystal structure (PDB code rev, RT/9-Cl-TIBO complex). Theprogram has the ability to define a radius of residues within a 5 Ådistance from the NNI molecule. As the modeling calculations progressed,the residues within the radius were allowed to move in accordance withthe energy minimization. Ten final docking positions were initiallychosen for each inhibitor modeling calculation but failed to reveal morethan two promising positions. Later, only two calculated positions wereset for the search target.

Calculations were carried out on a Silicon Graphics INIDIGO² using theCVFF force field in the Discover program and a Monte Carlo searchstrategy in Affinity (Luty, B. A. et al., J. Comp. Chem., 1995,16,454-464). No solvation procedures were used. Since the total numberof movable atoms exceeds 200, Conjugated Gradient minimization was usedinstead of the Newton minimization method. The initial coordinates ofthe compounds were generated using the Sketcher module within InsightII.Each final docking position was then evaluated by a score function inLUDI. The top scoring model was then compared with the composite bindingpocket and the known crystal structure of similar compounds and used forfurther analyses. The inhibitory constants (K_(i) values) of thepositioned NNI compounds were evaluated using the LUDI score function(Bohm, H. J., J. Comput. Aided Mol. Des., 1994, 8, 243-256; Bohm, H. J.,J. Comput Aided Mol. Des., 1992, 6, 593-606).

Several modifications were imposed during the calculation of inhibitoryconstants (K_(i) values) of the positioned compounds using the LUDIscore function (Bohm, H. J. 1994 supra; Bohm, H. J. 1992 supra). First,the molecular surface areas (MS) were directly calculated from thecoordinates of the compounds in docked conformations using the MSprogram. Second, the number of rotatable bonds (NR), which was assessedinaccurately by INSIGHTII (rigidity imposed by hydrogen bonding was notaccounted for in the program), was re-evaluated. Third, it was assumedthat the conserved hydrogen bond with RT was assumed to not deviatesignificantly from the ideal geometry. This assumption was supported bythe fact that in the known crystal structures of RT complexes, allhydrogen bonds between NNIs and RT are near the ideal geometry. Last,for the trovirdine compounds, an additional penalty was imposed for acharged group or halogen atoms when positioned near the ring plane of aprotein residue such as tryptophan 229 because the interaction was notadequately accounted for in the LUDI score. The working modification ofthe LUDI scoring function for the PETT compounds included subtracting ascore of P from the total LUDI score when the ring plane of the Trp229was within 5 Å from a para substituent (R):LUDI Score=MS*BS*2.93+85(H-bond)−NR*24.2−100−P; where

-   -   P=200, when R=a hydrophilic group, e.g. —OH or —NO2;    -   P=100, when R=a para-halogen atom, e.g. —F, —Cl or —Br;    -   P=50, when R=a para-methoxy, e.g. —OMe;    -   P=0, when R=a hydrophobic group, e.g. H, CH3;        Consequently, the K_(i) values for the modeled compounds were        more predictable than they would be without such modification        (Bohm, H. J. 1994 supra; Bohm, H. J. 1992 supra).        Contact Surface and Gap Analysis

Independent of the composite binding pocket and as a follow-up to thedocking procedure, computer programs were used to analyze the surfacecomplementarity between the compounds and the binding site residues.This analysis provided another useful way to examine bindinginteractions, based solely upon the structure that was used for docking(RT/9-Cl TIBO for PETT and RT/MKC-442 for DABO and HEPT) (Das, K. etal., J. Mol. Biol., 1996, 264, 1085-1100).

A number of computer programs were written to analyze the surface of thecompounds in the NNI binding site of RT and to better visualize anyspatial gaps between the compounds and nearby residues of the RTprotein. The algorithm used in these programs was based on a series ofcubic grids surrounding the compound, with a user-defined grid spacing.All cubes were coded based on the distance and the nature of theinteraction with the protein residues and/or compound atoms. The cubesthat overlap both protein and compound within the contact radius aredisplayed as spheres and were selected to represent the buried surface(user-defined contact radius was the van der Waals radius plus anuncertainty factor, dependent on the reliability of source coordinates).All other cubes that did not interact with protein residues and werewithin a certain distance from the compound were selected to representthe gap space (space unoccupied by compound or protein) and aredisplayed as rods.

A graphic interface was then used to examine whether the “gap” spherescould be connected with the compounds without intersecting the “contact”spheres. If the criterion was met, the points that stemmed from thesurface of the compound were defined as an expandable region (eligiblefor synthetic modification). The spheres generated by the programs(shown in FIG. 3) represent the sites buried by protein residues,indicating regions of the compound which are probably not available forderivatization.

FIG. 4 shows the binding pocket embellished with a grid of red rodswhich represent unoccupied space between the compound and active siteresidues, providing a complementary view to that shown by the spheres.The grid illustrates the candidate sites for derivatization of thecompound and, when used as a distance scale (the length of one rodrepresents 1 Å), also indicates the volume available for new functionalgroups.

One example of a useful program is the “SeeGap” program, whose code islisted below in Example 11, together with instructions for its use.

Composite NNI Binding Pocket of RT Reveals Protein Flexibility AndFuture Inhibitor Modification Sites

The integrated structural information surprisingly revealed a muchlarger binding site than any shown in individual structures and servedas a probe to define the potentially usable space in the binding site(FIG. 1). The three-dimensional binding site can be used as a referencepoint for the analysis of compounds which have been positioned by adocking procedure.

Upon inspection of the pocket it was apparent that although there are nolarge-scale conformational changes within the NNI binding site, a numberof RT protein residues in contact with the inhibitors are relativelyflexible and vary from structure to structure. These residues includeTyr180, Tyr181, Tyr318, Try319, Phe227, Leu234, Trp229, Pro95, andGlu138 (the latter from p51 subunit of RT).

As shown in FIG. 2B, the surface of the composite binding pocket whichis overlaid with the RT-TIBO binding site is a short distance (<1.5 Å)away from or even extends past RT residues 234-236, Y188, F227, and thebackbone of K101. This indicates that these residues are flexible andcan be displaced by the right substituent on an inhibitor.

The composite binding pocket of the invention, unlike a single crystalstructure, is able to integrate the nature and extent of the flexibilityof the active site residues in the NNI binding site of RT. This uniquelypermits prediction of potential modification sites on PETT, DABO, andHEPT derivatives after positioning the compounds in the NNI active siteof RT. The method for designing new NNI compounds was particularlyuseful given the fact that no known crystal structures exist for RT-PETTand RT-DABO complexes, a fact which in this case would prevent thesuccessful application of traditional structure-based drug designmethods. Importantly, the model was validated by experimentallydemonstrating the superior potency of newly designed agents, predictedto have strong RT inhibitory activity, based upon the low K_(i) valuesestimated.

Example 2 Predicted Efficacy of HEPT Derivatives

Compounds listed in Table 1 have been modeled into the NNI binding siteof RT (RT/MKC 422 complex) using the docking procedure. The modeledpositions were compared with the composite binding pocket of theinvention, having the coordinates set forth in Table 9. Modeling wasfollowed by analysis with the LUDI score function.

All of the positions of the compounds with top scores fall into thebutterfly-shaped binding site, with the benzyl ring residing in wing 1and the thymine ring in the wing 2 (FIG. 2). For all compounds tested,the benzyl ring is near Trp229 and the N−1 group is near Pro236, atypical position observed in crystal structures (FIG. 1B). The trend ofcalculated values listed in Table 1 shows that the K_(i) value decreasesas a result of three factors: para substituents (R2) removed from thebenzyl ring, larger alkyl groups added to the thymine ring (R₁), andsulfur atoms substituted for oxygen (at X and/or Y). The modelingcalculations, along with the application of the composite NNI bindingpocket, provided a guideline for the synthesis of lead compoundsdesigned to have potent anti-HIV activity. The choice of compounds wasalso based on synthetic feasibility. TABLE 1 Results of modelingcalculations for HEPT derivatives

Accessible Molecular Buried LUDI LUDI Surface surface Surface ScoreScore^(d) Ki^(d) X Y R₁ R₂ R₃ NR^(a) (Å²) (Å²) (%) (Lipo) (Sum) (μM) O OEt F Et 6 549 296 n.d. n.d. n.d. n.d. O O Et Br Et 6 576 311 n.d. n.d.n.d. n.d. S O Me OMe Et 6 558 303 n.d. n.d. n.d. n.d. O O Me H Et 5 505269 85 599 463 23 O O Et H Et 6 528 284 87 661 501 9.8 O O i-Pr H Et 6541 294 88 688 528 5.2 S O Me H Et 5 512 275 87 703 567 2.1 S O Et H Et6 536 290 90 732 572 1.9 S O i-Pr H Et 6 550 300 89 741 580 1.5 S S Me HEt 5 521 283 86 706 570 2.0 S S Et H Et 6 545 297 90 756 595 1.1 S Si-Pr H Et 6 557 308 90 777 617 0.68 S S Me H Me 4 491 266 84 661 549 3.2S S Et H Me 5 514 280 88 703 567 2.1 S S i-Pr H Me 5 527 290 90 738 6020.95Me = methyl, Et = ethyl, i-Pr = isopropyln.d. (not determined) means high K_(i) values resulting fromenergetically unfavorable rotation of Trp229 which sterically hindersbinding in cases of the para substitution, as revealed by modeling.^(a)NR = number of rotatable bonds in the compound. Used in the LUDIcalculation to reflect the loss of binding energy due to freezing ofinternal degrees of freedom.^(b)Molecular surface area calculated using the program GRASP, anddefined as the boundary of the volume within any probe sphere (meant torepresent a water molecule) of given radius sharing no volume with thehard sphere atoms which make up the molecule. The values are slightlysmaller than the ones approximated by LUDI program. The accessiblesurface can be defined as the locus of the centers of all possible suchprobes in contact with the hard sphere atoms.# Alternatively it can be defined as the hard sphere surface if eachatomic radius is increased by the probe radius (1.4 Å radius).^(c)Buried surface represents the percentage of molecular surface incontact with the protein calculated by LUDI based on the dockedpositions. Based on published crystal structures of RT complexes, thecalculation shows that these values could be as low as 77% (in RT/HEPTcomplex) and can be as high as 90% (in RT/APA complex) but most of themincluding RT/MKC average around 84%. Therefore, the calculated valuesmay be in the worst case slightly overestimated.^(d)Ideal hydrogen bond distances and angles between the compounds andthe protein are assumed in all cases for K_(i) and Score (sum)calculation. In published crystal structures of RT complexes, hydrogenbond geometry's are indeed close to ideal; the amide carbonyl of residueA101 on a loop demonstrates a substantial flexibility which canaccommodate the best geometry for hydrogen bonding.Synthesis of HEPT Derivatives

The compounds listed in Table 1 above can be synthesized by reaction ofsubstituted aryl acetonitriles and appropriately functionalized 2-bromoethyl esters, for example in the presence of zinc in refluxingtetrahydrofuran. Products of the reaction are purified by gelchromatography. Generated 3-oxo esters are next converted into5-alkyl-6-(arylmethyl)-2-thiouracils with chloroacetic acid, e.g.,overnight to yield 5-alkyl-6-(arylmethyl)uracils. The final step in thesynthesis is reaction of the uracil with hexamethyldisilaaane (HMDS) inthe presence of ammonium sulfate. Subsequent treatment with acetals andtrimethyl silyl triflate in acetonitrile leads to the formation ofN-substituted uracil and thiouracil derivatives.

These and other known methods can be used to synthesize the compounds ofthe invention.

Example 3 DABO Derivatives

Chemical Synthesis

All chemicals were used as received from Aldrich Chemical Company(Milwaukee, Wis.). All reactions were carried out under nitrogen. Columnchromatography was performed using EM Science silica gel 60 and one ofthe following solvents: ethyl acetate, methanol, chloroform, hexane, ormethylene chloride. Nuclear magnetic resonance (NMR) spectra wererecorded on a Varian (Palo Alto, Calif.) 300 MHz instrument (Mercury2000 model) and chemical shifts (δ) are reported in parts per million(ppm) relative to tetramethylsilane as an internal standard at 0 ppm.¹³C NMR spectra were recorded at 75 MHz in CDCl₃ on the same instrumentusing a proton decoupling technique. The chemical shifts reported for¹³C NMR are referenced to the chloroform triplet at 77 ppm. Meltingpoints were measured using a Mel-Temp 3.0 (Laboratory Devices Inc.,Holliston, Mass.) melting apparatus and are uncorrected. UV spectra wererecorded from a Beckmann (Fullerton, Calif.) model DU 7400 UV/Visspectrometer using a cell path length of 1 cm and methanol solvent.Fourier Transform Infrared spectra were recorded using an FT-Nicolet(Madison, Wis.) model Protege 460 instrument. Mass spectrum analysis wasperformed using a Hewlett-Packard (Palo Alto, Calif.) Matrix AssistedLaser Description time-of-flight (MALDI-TOF) spectrometer (model G2025A)in the molecular ion detection mode (matrix used wascyanohydroxycinnamic acid). Some samples were analyzed using a Finnigan(Madison, Wis.) MAT 95 instrument. Elemental analysis was performed byAtlantic Microlabs (Norcross, Ga.).

General Procedure for the Synthesis of DABO Compounds 3a-d:

The 5-alkyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-onederivatives 3a-d were prepared as shown in Scheme 1. Scheme 3

1-3 R₁ R₂ a H Me b H Et c H i-Pr d Me i-Pr

-   -   Reagents and conditions: a) R₂CHBrCOOEt/Zn/THF, b) HCl(aq), c)        (H₂N)₂CS/Na/EtOH, d) DMF, K₂CO₃, Chloromethyl methyl sulfide,        15h.

Ethyl-2-alkyl-4-(phenyl)-3-oxobutyrates 1a-d were obtained fromcommercially available phenyl acetonitrile. The 0-ketoesters werecondensed with thiourea in the presence of sodium ethoxide to furnishthe corresponding thiouracils 2a-d. Compounds (1 a-d and 2 a-d) wereproduced by a methods previously described (Danel, K. et al., ActaChemica Scandinavica, 1997, 51, 426-430; Mai, A. et al., J. Med. Chem.,1997, 40, 1447-1454; Danel, K. et al., J. Med. Chem., 1998, 41,191-198).

Subsequent reaction of thiouracil with methylchloromethyl sulfide inN,N-dimethylformamide (DMF) in the presence of potassium carbonateafforded compounds 3a-d in moderate yields A mixture of thiouracilcompound 2 (1 mmol), methylchloromethyl sulfide (1 mmol), and potassiumcarbonate (1 mmol) in anhydrous DMF (5 ml) was stirred overnight at roomtemperature. After treatment with water (50 ml), the solution wasextracted with ethyl acetate (3×50 ml). The combined extracts werewashed with saturated NaCl (2×50 ml), dried (MgSO₄), filtered andconcentrated in vacuo to give the crude products 3a-d which werepurified by column chromatography (hexane:ethyl acetate eluent).

X-Ray Crystallography

Yellow rectangular plates of compound 3b were grown from tetrahydrofuranby slow evaporation at room temperature. X-ray diffraction data for a0.5×0.2×0.08 mm plate crystal of compound 3b was collected at roomtemperature using a SMART CCD X-ray detector (Bruker Analytical X-raySystems, Madison, Wis.). Structure solution and refinement was performedusing the SHELXTL suite of programs (Bruker Analytical X-ray Systems,Madison, Wis.). All nonhydrogen atoms were refined using anisotropicdisplacement parameters. Hydrogen atoms were placed at ideal positionsand refined as riding atoms with relative isotropic displacementparameters.

The refined small molecule X-ray crystal structure of compound 3b isshown as an Oak Ridge Thermal Ellipsoid Program (ORTEP) drawing in FIG.8. Table 2 lists the crystal data and structure refinement statisticsfor compound 3b. Data was collected at room temperature (λ=0.71073 Å),refined using full-matrix least-squares refinement on F², and correctedfor absorption using semi-empirical psi-scans. TABLE 2 Unit Cell a =4.7893(4) Å b = 10.8709(10) Å c = 30.040(3) Å α = 90° β = 92.474(2)° γ =90° Space Group P2₁/n Unit Cell Volume 1562.5(2) Å³ Z 4 θ range for datacollection 1.36 to 28.27° Limiting indices −6 ² h ² 6 −8 ² k ² 14 −39 ²l ² 37 Reflections collected 8744 Independent reflections 3507 (R_(int)= 0.0486) Data/restraints/parameters 3507/0/183 Goodness-of-fit on F²1.095 Final R indices [I>2σ(I)] R1 = 0.0666, wR2 = 0.1384 R indices (alldata) R1 = 0.1114, wR2 = 0.1569 Absorption coefficient 0.338 mm⁻¹ Max.and min. transmission 0.8356 and 0.6542 Extinction coefficient0.0004(11) Largest difference peaks 0.279 and −0.211 eÅ⁻³R_(int) = Σ|F_(o) ² − <F_(o) ²>|/Σ|F_(o) ²|, R1 = Σ||F_(o)| −|F_(c)||/Σ|F_(o)|wR2 = {Σ[w(F_(o) ² − F_(c) ²)²]/Σ[w(F_(o) ²)²]}^(1/2)GooF = S = {Σ[w(F_(o) ² − F_(c) ²)²]/(n − p)}^(1/2), where n =reflections, p = parametersPhysical Data of Synthesized Compounds:

5-methyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-1H-one (3a)

Yield 62%; mp 148-149° C.; ¹H NMR(CDCl₃): δ 2.10 (s, 3H), 2.14 (s, 3H),3.91 (s, 2H), 4.29 (s, 2H), 7.29-7.26 (m, 5H), 12.20 (s, 1H); ¹³CNMR(CDCl₃): δ 10.7 (CH₃), 15.5 (SCH₃), 36.6 (CH₂Ph),41.0 (SCH₂), 116.7(C-5), 137.6-126.4 (Ph), 155.2 (C-6), 162.0 (C-4), 165.1 (C-2); Cl-MS:293.1 (M+1).

5-ethyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-1H-one (3b)

Yield 65%; mp 124-126° C.; ¹H NMR(CDCl₃): δ 1.08 (t, 3H), 2.12 (s, 3H),2.58 (q, 2H), 3.91 (s, 2H), 4.26 (s, 2H), 7.28-7.26 (m, 5H), 12.30 (s,1H); ¹³C NMR(CDCl₃): δ 13.1 (CH₃), 15.4 (SCH₃), 18.7 (CH₂), 36.4(CH₂Ph), 40.3 (SCH₂), 122.4 (C-5), 138.0-126.3 (Ph), 155.4 (C-6), 161.5(C-4), 165.2 (C-2); Cl-MS: 307.1 (M+1).

5-isopropyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-1H-one (3c)

Yield 57%; mp 116-117° C.; ¹H NMR(CDCl₃): δ 1.22 (d, 6H), 2.07 (s, 3H),3.03 (q, 1H), 3.88 (s, 2H), 4.21 (s, 2H), 7.24-7.13 (m, 5H), 12.43 (s,1H); ¹³C NMR(CDCl₃): δ 15.4 (SCH₃), 19.6 (CH₃), 28.0 (CH), 36.3 (CH₂Ph),40.9 (SCH₂), 125.3 (C-5), 138.3-126.3 (Ph), 155.5 (C-6), 161.1 (C-4),164.5 (C-2); CI-MS 321.1 (M+1).

5-isopropyl-2-[(methylthiomethyl)thio]-6-(3,5-dimethylbenzyl)-pyrimidin-4-1H-one(3d)

Yield 67%; mp 116-120° C.; ¹H NMR(CDCl₃): δ 1.28 (d, 6H), 2.15 (s, 3H),2.27 (s, 6H), 3.10 (q, 1H), 3.88 (s, 2H), 4.31 (s, 2H), 6.84 (s, 3H),12.42 (s, 1H); ¹³C NMR(CDCl₃): δ 15.3 (SCH₃), 19.6 (CH₃), 21.2 (CH₃),28.0 (CH), 36.3 (CH₂Ph), 40.8 (SCH₂), 125.2 (C-5), 138.0-126.5 (Ph),155.4 (C-6), 161.3 (C-4), 164.7 (C-2); Cl-MS: 349.2 (M+1).

Modeling and Design of DABO Compounds:

The calculated molecular coordinates of DABO compounds which wereenergy-minimized and docked into the NNI binding site adopted aconformation remarkably similar to that of the crystal structure ofcompound 3b. FIG. 7B shows the modeled coordinates superimposed on thecrystal structure coordinates of 3b and illustrates their conformationalsimilarity, suggesting that the final docked positions of the DABOcompounds in the NNI pocket were energetically favorable and quitesuitable for these studies. Multiple sterically allowed unoccupiedspatial gaps in the binding site were identified from the dockingstudies which could be filled by strategically designed functionalgroups (FIG. 7B).

The docked DABO molecule (compound 3a) unexpectedly showed significantspace surrounding the benzyl ring and the 5th position of the thyminering, which led to design of compounds 3b, 3c and 3d. The inhibitionconstants of the docked molecules were calculated based on a LUDI scorefunction and are listed in Table 3. The calculated K_(i) valuessuggested that compounds 3c and 3d would be particularly activeinhibitors of RT.

Compound 3d, which differs from compound 3c by the addition of twomethyl groups to the benzyl ring, provides more hydrophobic contact withthe NNI binding pocket and was predicted to be more potent than compound3c, based on the modeling studies. Calculations indicate that compounds3a-3d have progressively larger molecular surface areas but stillmaintain approximately the same percentage of the molecular surface areain contact with the protein residues. Consequently, the calculatedcontact surface area between the protein and the compound increases inthe following order: compound 3a, 3b, 3c, and 3d. This increased surfacearea in turn dictates a decrease in calculated K_(i) values, with 3ahaving the worst value and 3d the best.

The Tyr183 residue of the HIV RT is located in the catalytic regionwhich has a conserved YMDD motif characteristic of reversetranscriptases. Therefore, the displacement of this tyrosine residue caninterfere with catalysis and render the HIV-1 RT protein inactive. Bulkysubstituents at the 5th position of the thymine ring could indirectlyaccomplish such inactivation by displacing Tyr181 which is near Tyr183(Ding, J. et al., Nat. Struct. Biol., 1995, 2, 407-415). The compositebinding pocket shows sufficient room for at least a 3-carbon group atthe 5th position. The addition of a methyl, ethyl or isopropyl group atthe 5th position of the thymine ring is expected to lead to higheraffinity for the relatively hydrophobic environment at this location ofthe binding pocket. The favorable hydrophobic contact increases as thehydrophobic group at the 5th position gets bulkier. As the DABOderivative binds to the site, the ethyl or isopropyl group can alsocause the nearby Tyr181 residue to rotate away from the inhibitor.

Modeling studies showed that this change in conformation in turn affectsthe positions of neighboring Tyr183 and Tyr188 which may contribute tothe inactivation of HIV-1 RT. The benzyl ring of compounds 3a-3d islocated near a region surrounded by the hydrophobic ring planes ofresidues Trp229, Pro95, Y188 and Y181. The analysis of compounds 3a-3cin the composite binding pocket suggests that the benzyl ring would belocated on the boundary of the pocket, near residue Y188. A parasubstituent of the ring is situated perpendicular to the ring plane ofnearby Trp229, within van der Waals contact, and leaves a lot of spaceunfilled between the compound and Pro95. With a slight conformationalrotation of the benzyl ring, compound 3d, with the addition of twomethyl groups, was found to better fill the composite binding pocket(FIG. 7B). Such observations indicate that further modifications to thebenzyl ring could lead to even more potent inhibitors. TABLE 3 DaboCompounds

Compound Ludi^(a) Number R₁ R₂ M.S.^(b)(Å²) B.S.^(c)(%) Lipo ScoreK_(i)(μM) 3a H Me 275 88 709 3.3 3b H Et 283 88 730 2.0 3c H i-Pr 301 89785 0.56 3d Me i-Pr 329 89 875 0.05^(a)Ludi K_(i) values were calculated based on the empirical scorefunction in Ludi program (Bohm, H. J., J. Comput. Aided. Mol. Des.,1994, 8, 243-256; 1996). Ideal hydrogen bond distances and anglesbetween compounds and protein are assumed in all cases for Ludi K^(i)and Ludi Score calculation. In published crystal structures of RTcomplexes, hydrogen bond geometries are indeed close to ideal; the amidecarbonyl of residue A101 on a loop demonstrates substantial# flexibility which can accommodate the best geometry for hydrogenbonding. The number of rotatable bonds (=2) is used in the Ludicalculation to reflect the loss of binding energy due to freezing ofinternal degrees of freedom.^(b)MS, molecular surface area calculated using Connolly's MSprogram(Connolly, M. L., Science, 1983, 221, 709-713). Defined asboundary of volume within any probe sphere (meant to represent a watermolecule) of given radius sharing no volume with hard sphere atoms whichmake up the molecule. Values are slightly smaller than thoseapproximated by Ludi program.^(c)BS, buried surface: percentage of molecular surface in contact withprotein calculated by Ludi relative to docked positions. Based onpublished crystal structures of RT complexes, the calculation shows thatthese values could be as low as 77% (in RT-HEPT complex) and can be ashigh as 90% (in RT-APA complex) but most of them including RT-MKCaverage around 84%.Predictable Activities

The trend of the calculated K_(i) values based on the modeling and onthe use of the composite binding pocket, with surprising accuracy,predicted the trend of the experimentally determined IC₅₀ values fromHIV replication assays. Compounds 3a-3d were tested for RT inhibitoryactivity in cell-free assays using purified recombinant HIV RT (listedas IC₅₀[rRT] in Table 4), as well as by in vitro assays of anti-FIVactivity in HTLV_(IIIB)-infected peripheral blood mononuclear cells(IC₅₀[p24] in Table 4) (Zarling, J. M. et al., Nature, 1990, 347, 92-95;Erice, A. et al., Antimicrob. Ag. Chemother., 1993, 37, 835; Uckun, F.M. et al., Antimicrobial Agents and Chemotherapy, 1998, 42, 383).

Larger compounds which better fill the composite binding pocket and havelower calculated K_(i) values showed better IC₅₀[rRT] values. This isreflected by the enhancement of the inhibitory activity with theaddition of progressively larger groups such as methyl (3a), ethyl (3b),and isopropyl (3c) at the C-5 position of the thymine ring (see Table4). The same trend was also observed for IC₅₀[p24] values.

The lead DABO derivative,5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one(compound 3c), elicited potent anti-HIV activity with an IC₅₀ value lessthan 1 nM for inhibition of HIV replication (measured by p24 productionin HIV-infected human peripheral blood mononuclear cells) and showed nodetectable cytotoxicity (inhibition of cellular proliferation was >100μM as measured by MTA) (Table 4). In contrast to all previouslypublished data for DABO and S-DABO derivatives which were less activethan AZT and MKC-442 (Danel, K. et al., Acta Chemica Scandinavica, 1997,51, 426-430; Mai, A. et al., J. Med. Chem., 1997, 40, 1447-1454; Danel,K. et al., J. Med. Chem., 1998, 41, 191-198) and showed selectivityindices of <1,000, the novel compound 3c was more than 4-fold moreactive than AZT and MKC-442, and abrogated HIV replication in peripheralblood mononuclear cells at nanomolar concentrations with anunprecedented selectivity index (=IC₅₀[MTA]/IC₅₀[p24]) of >100,000.

The X-ray crystal structure of 3b was determined to compare itsconformation to that of the compound after docking into the NNI bindingsite. The refined small molecule X-ray crystal structure of compound 3bis represented as an ORTEP drawing in FIG. 8. The calculated molecularcoordinates of DABO compounds which were energy-minimized and dockedinto the NNI binding site adopted a conformation remarkably similar tothat of the crystal structure of compound 3b. FIG. 7B shows the modeledcoordinates superimposed on the crystal structure coordinates of 3b andillustrates their conformational similarity, suggesting that the finaldocked positions of the DABO compounds in the NNI pocket wereenergetically favorable. TABLE 4 Inhibitory Activity of DABO Compounds:

CC₅₀ Compound IC₅₀[rRT] IC₅₀[p24] [MTA] Number R₁ R₂ (μM) (μM) (μM)S.I.^(d) 3a H Me 18.8 4.5 >100    >22 3b H Et 9.7 0.8 >100    >125 3c Hi-Pr 6.1 <0.001 >100 >100,000 3d Me i-Pr 4.8 n.d. n.d. n.d. AZT >1000.04    50     1250 MKC-442 0.004 >100  >25,000^(d)Selectivity Index is equal to the ratio of fifty percent cytotoxicconcentration to IC₅₀.n.d. = not determined

Example 4 Synthesis of PETT Derivatives

Chemical Synthesis

All chemicals were used as received from Aldrich Chemical Company(Milwaukee, Wis.). All reactions were carried out under nitrogen. Columnchromatography was performed using EM Science silica gel 60 and one ofthe following solvents: ethyl acetate, methanol, chloroform, hexane, ormethylene chloride. Nuclear magnetic resonance (NMR) spectra wererecorded on a Varian (Palo Alto, Calif.) 300 MHz instrument (Mercury2000 model) and chemical shifts (δ) are reported in parts per million(ppm) relative to tetramethylsilane as an internal standard at 0 ppm.¹³C NMR spectra were recorded at 75 MHz in CDCl₃ on the same instrumentusing a proton decoupling technique. The chemical shifts reported for¹³C NMR are referenced to the chloroform triplet at 77 ppm. Meltingpoints were measured using a Mel-Temp 3.0 (Laboratory Devices Inc.,Holliston, Mass.) melting apparatus and are uncorrected. UV spectra wererecorded from a Beckmann (Fullerton, Calif.) model DU 7400 UV/Visspectrometer using a cell path length of 1 cm and methanol solvent.Fourier Transform Infrared spectra were recorded using an FT-Nicolet(Madison, Wis.) model Protege 460 instrument. Mass spectrum analysis wasperformed using a Hewlett-Packard (Palo Alto, Calif.) Matrix AssistedLaser Desorption time-of-flight (MALDI-TOF) spectrometer (model G2025A)in the molecular ion detection mode (matrix used wascyanohydroxycinnamic acid). Some samples were analyzed using a Finnigan(Madison, Wis.) MAT 95 instrument. Elemental analysis was performed byAtlantic Microlabs (Norcross, Ga.).

General Procedure for Synthesis of PETT Derivatives

Compounds I-1, I-3, and 1-4 were synthesized as described in Scheme 3.Trovirdine (1-2) was synthesized according to the literature procedure(Bell, F. W., et al., J. Med. Chem., 1995, 38, 4929-4936).

Physical Data of Synthesized Compounds:

N-[2-(2-pyridylethyl)]-N′-[2-(pyridyl)]-thiourea (I-1)

white solid (1 g, 49%); mp 98-100° C.; UV(MeOH) λmax: 293, 265, 247 and209 nm; IR(KBr Disc) ν 3415, 3222, 3050, 2360, 1600, 1533, 1479, 1436,1315, 1240, 1151 and 775 cm⁻¹; ¹H NMR (CDCl₃) δ 11.90 (s, 1H), 8.8 (s,1H), 8.60-8.58 (d, 1H), 8.03-8.01 (d, 1H), 7.65-7.56 (m, 2H), 7.27-7.14(m, 2H), 6.93-6.89 (d, 1H), 6.80-6.77 (d, 1H) 4.23-4.15 (q, 2H) and3.41-3.20 (t, 2H); ¹³C NMR(CDCl₃) δ 179.2, 158.9, 153.0, 149.2, 145.5,138.5, 136.4, 123.5, 121.4, 117.7, 111.8, 44.9, and 36.9; MALDI-TOF massfound, 257.1(M−1), calculated, 258.3; Anal. (C₁₃H₁₄N₄S) C, H, N, S.

N-[2-(1-piperidinoethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (I-3)

white solid (2 g, 74%); mp 150-152° C.; UV (MeOH) λmax: 306, 275 and 205nm; IR(KBr Disc) ν 3155, 3077, 2935, 2850, 2360, 1591, 1525, 1465, 1319,1226, 1095, 827 and 756 cm⁻¹; ¹H NMR (CDCl₃) δ 11.53 (br s, 1H), 9.72(br s, 1H), 8.22 (d, 1H), 7.72-7.68 (dd, 1H), 6.95-6.92 (d, 1H),3.84-3.78 (q, 2H), 2.61-2.57 (t, 2H), 2.45 (br s, 4H), 1.64-1.48 (m,6H); ¹³C NMR(CDCl₃) δ 178.1, 151.8, 146.3, 140.8, 113.5, 112.6, 56.1,54.0, 43.0, 26.3, and 24.3, MALDI-TOF mass found, 343.5, calculated,343.3; Anal. (C₁₃H₁₉BrN₄S)C, H, N, S, Br.

N-[2-(2,5-dimethoxyphenylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (I-4)

white solid (2 g, 67%); mp 133-138° C.; UV (MeOH) λmax: 202, 205, 231,276 and 300 nm; IR(KBr Disc) ν 3209, 3152, 3078, 3028, 2951, 2831, 1595,1533, 1468, 1306, 1227, 1095, 1059, 1022, 862, 825, 796, 707 cm⁻¹;¹HNMR(CDCl₃) δ 11.24 (br s, 1H), 9.30 (br s, 1H), 8.10-8.09 (d, 1H),7.65 (dd, 1H), 6.82-6.76 (m, 4H), 4.03-3.97 (q, 2H), 3.77 (s, 3H), 3.76(s, 3H), 3.00-2.96 (t, 2H); ¹³C NMR(CDCl₃) 5178.7, 153.1, 151.8, 151.7,146.5, 140.9, 128.1, 117.7, 113.3, 112.6, 111.2, 110.9, 55.7, 55.5,45.6, and 29.9; MALDI-TOF mass found, 394.0 (M−1), 396.0 (M+1),calculated, 395.0; Anal. (C₁₆H₁₈BrN₃O₂S)C, H, N, S, Br.

Chemical Synthesis II

Compounds II-1-9 were synthesized according to Scheme 4. In brief,2-amino-5-bromopyridine was condensed with 1, 1-thiocarbonyl diimidazoleto furnish the precursor thiocarbonyl derivative (A). Further reactionwith appropriately substituted phenylethyl amine gave the target PETTderivatives in good yields.

General Procedure for Synthesis

Thiocarbonyldiimidazole (8.90 g, 50 mmol) and 2-amino-5-bromo pyridine(8.92 g, 50 mmol) were added to 50 mL of dry acetonitrile at roomtemperature. The reaction mixture was stirred for 12 h and theprecipitate filtered, washed with cold acetonitrile (2×25 mL), and driedunder vacuum to afford (11.40 g, 80%) of compound A. To a suspension ofcompound A (0.55 eqv) in dimethyl formamide (15 mL) an appropriate amine(0.50 eqv) was added. The reaction mixture was heated to 100° C. andstirred for 15 hours. The reaction mixture was poured into ice-coldwater and the suspension was stirred for 30 minutes. The product wasfiltered, washed with water, dried, and further purified by columnchromatography to furnish the target compounds 1-9 in good yields.

Physical Data of Synthesized Compounds:

N-[2-(2-methoxyphenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-1)

yield: 65%; mp 143-145° C.; UV (MeOH) λmax: 202, 205, 275 and 306 nm;IR(KBr) ν 3211, 3153, 3036, 2956, 2835, 1593, 1533, 1462, 1242, 1186,1036, 1007, 862, 812, 756, 708 cm⁻¹; ¹H NMR (CDCl₃) δ 11.22 (br s, 1H),9.37 (br s, 1H), 8.02-8.01 (d, 1H), 7.69-7.65 (dd, 1H), 7.28-7.18 (m,2H), 6.94-6.80 (m, 3H), 4.04-3.98 (q, 2H), 3.81 (s, 3H), 3.04-2.99 (t,2H); ¹³C NMR(CDCl₃) 8178.7, 157.6, 151.7, 146.3, 141.0, 130.7, 127.9,126.8, 120.3, 113.5, 112.5, 110.3, 55.2, 45.6, 29.8; Maldi Tof found:366.0 (M+1), calculated: 365.0; Anal. (C₁₅H₁₆BrN₃OS) C, H, N, S.

N-[2-(2-fluorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-2)

yield: 71%; mp 156-157° C.; UV (MeOH) λmax: 209, 256, 274 and 305 nm;IR(KBr) ν 3446, 3234, 3163, 3055, 2935, 1672, 1595, 1560, 1531, 1466,1390, 1362, 1311, 1265, 1227, 1169, 1136, 1089, 1003, 864, 825, 756cm⁻¹; ¹H NMR (CDCl₃) δ 11.36 (br s, 1H), 9.47 (br s, 1H), 8.05-8.04 (d,1H), 7.72-7.68 (dd, 1H), 7.30-7.03 (m, 4H), 6.87-6.84 (d, 1H), 4.06-3.99(q, 2H), 3.10-3.05 (t, 2H); ¹³C NMR(CDCl₃) δ 179.1, 163.1, 151.7, 146.2,141.1, 131.2, 131.1, 128.5, 128.4, 124.1, 115.5, 115.2, 113.6, 112.2,45.8 and 28.2; ¹⁹F NMR(CDCl₃) 8-42.58 &-42.55 (d); Maldi Tof found:355.0 (M+1), calculated: 354.0; Anal. (C₁₄H₁₃BrFN₃S) C, H, N, S.

N-[2-(2-chlorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-3)

yield: 72%; mp 137-139° C.; UV (MeOH) λmax: 208, 213, 256, 275 and 305nm; IR(KBr) ν 3433, 3221, 3157, 3089, 3037, 2922, 2866, 1668, 1597,1535, 1466, 1338, 1263, 1209, 1188, 1130, 1095, 1053, 1001, 864, 823,750 cm⁻¹; ¹H NMR (CDCl₃) δ 11.41 (br s, 1H), 9.54 (br s, 1H), 8.17-8.16(d, 1H), 7.83-7.79 (dd, 1H), 7.50-7.30 (m, 4H), 6.97-6.94 (d, 1H),4.19-4.13 (q, 2H), 3.30-3.26 (t, 2H); ¹³C NMR(CDCl₃) δ 179.2, 151.7,146.3, 141.2, 136.3, 134.2, 131.1, 129.6, 128.1, 126.8, 113.6, 112.7,45.2, and 32.5; Maldi Tof found: 371.8 (M+1), calculated: 371.0; Anal.(C₁₄H₁₃BrClN₃S)C, H, N, S, Br.

N-[2-(3-methoxyphenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-4)

yield: 68%; mp 155-156° C.; V (MeOH) λmax: 208, 274 and 306 nm; IR(KBr)ν 3454, 3236, 3147, 3030, 2951, 2869, 2827, 1591, 1545, 1525, 1466,1304, 1265, 1229, 1188, 1151, 1095, 1051, 1024, 980, 860, 825, 789, 698cm⁻¹; ¹H NMR (CDCl₃) δ 11.30 (br s, 1H), 9.25 (br s, 1H), 8.05-8.04 (d,1H), 7.71-7.67 (dd, 1H), 7.29-7.24 (t, 1H), 6.89-6.78 (m, 4H), 4.05-3.99(q, 2H), 3.81 (s, 3H), 3.00-2.96 (t, 2H); ¹³C NMR(CDCl₃) δ 178.9, 159.7,151.6, 146.4, 141.1, 140.3, 129.6, 121.2, 115.0, 113.4, 112.7, 111.6,55.1, 47.1 and 34.8; Maldi Tof found: 367.0 (M+2), calculated: 365.0;Anal. (C₁₅H₁₆BrN₃OS)C, H, N, S.

N-[2-(3-fluorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-5)

yield: 73%; mp 171-172° C.; UV (MeOH) λmax: 202, 208, 258, 275 and 306nm; IR(KBr) v 3213, 3155, 3084, 3028, 2866, 1595, 1533, 1477, 1336,1308, 1229, 1211, 1173, 1136, 1092, 1026, 935, 870, 827, 791, 740 cm⁻¹;¹H NMR (CDCl₃) 611.33 (br s, 1H), 9.46 (br s, 1H), 8.05-8.04 (d, 1H),7.73-7.69 (dd, 1H), 7.31-7.26 (m, 1H), 7.08-6.97 (m, 3H), 6.87-6.83 (d,1H), 4.06-3.99 (q, 2H), 3.05-3.00 (t, 2H); ¹³C NMR (CDCl₃) δ 179.1,163.1, 151.7, 146.2, 141.2, 130.1, 129.9, 124.5, 115.9, 115.6, 113.7,113.5, 113.4, 112.8, 46.7 and 34.6; ¹⁹F NMR(CDCl₃) δ −37.30 &−37.33 (d);Maldi Tof found: 354.0 (M+), calculated: 354.0; Anal. (C₁₄H₁₃BrFN₃S) C,H, N, S.

N-[2-(3-chlorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-6)

yield: 72%; mp 163-165° C.; V (MeOH) λmax: 202, 213, 258, 276 and 305nm; IR(KBr) v 3242, 3161, 3043, 2929, 1593, 1579, 1547, 1527, 1466,1313, 1227, 1167, 1095, 997, 889, 827, 812, 785, 700 cm⁻¹; ¹H NMR(CDCl₃) δ 11.33 (br s, 1H), 9.37 (br s, 1H), 8.09-8.08 (d, 1H),7.73-7.69 (dd, 1H), 7.28-7.15 (m, 4H), 6.85-6.82 (d, 1H), 4.04-3.98 (q,2H), 3.02-2.97 (t, 2H), ¹³C NMR (CDCl₃) δ 179.1, 151.6, 146.3, 141.2,140.7, 134.2, 129.8, 129.0, 127.0, 126.8, 113.4, 112.8, 46.7 and 34.5;Maldi Tof found: 371.8 (M+1), calculated: 371.0; Anal. (C₁₄H₁₃BrClN₃S)C, H, N, S.

N-[2-(4-methoxyphenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-7)

yield: 85%; mp 178-179° C.; UV (MeOH) λmax: 205, 226, 275 and 305 mm;IR(KBr) ν 3221, 3159, 3042, 2931, 2827, 1587, 1510, 1464, 1311, 1225,1165, 1088, 1034, 820, 773, 708 cm⁻¹; ¹H NMR (CDCl₃) δ 11.30 (br s, 1H),9.87 (br s, 1H), 8.00-7.99 (d, 1H), 7.67-7.63 (dd, 1H), 7.21-7.18 (d,2H), 6.95-6.85 (m, 3H), 4.00-3.93 (q, 2H), 3.81 (s, 3H), 2.96-2.92 (t,2H); ¹³C NMR (CDCl₃) δ 179.1, 158.0, 151.9, 145.8, 140.7, 130.6, 129.6,113.8, 113.7, 112.1, 55.1, 46.9 and 33.8; Maldi Tof found: 366.0 (M+1),calculated: 365.0; Anal. (C₁₅H₁₆BrN₃OS) C, H, N, S.

N-[2-(4-fluorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-8)

yield: 69%; mp 177-178° C.; UV (MeOH) λmax: 208, 211, 274 and 306 nm;IR(KBr) ν 3456, 3213, 3155, 3086, 3028, 2868, 1595, 1560, 1533, 1477,1336, 1308, 1238, 1211, 1173, 1136, 1092, 1026, 933, 869, 827, 791, 741,694 cm⁻¹; ¹H NMR (CDCl₃) δ 11.29 (br s, 1H), 9.27 (br s, 1H), 8.04-8.03(d, 1H), 7.73-7.69 (dd, 1H), 7.27-7.22 (m, 2H), 7.04-6.99 (m, 2H),6.83-6.79 (d, 1H), 4.03-3.96 (q, 2H), 3.02-2.97 (t, 2H); ¹³C NMR(CDCl₃)δ 179.1, 163.2, 151.6, 146.3, 141.2, 134.3, 130.3, 130.2, 115.4, 115.2,113.5, 112, 47.0, and 34.1; ¹⁹F NMR (CDCl₃) 6-40.55 (m); Maldi Toffound: 354.8 (M+1), calculated: 354.0; Anal. (C₁₄H₁₃BrFN₃S) C, H, N, S.

N-[2-(4-chlorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-9)

yield: 71%; mp 180-183° C.; UV (MeOH) λmax: 206, 209, 219, 256, 275 and305 nm; IR(KBr) ν 3221, 3153, 3086, 3022, 2931, 1674, 1593, 1562, 1533,1473, 1406, 1340, 1304, 1265, 1227, 1169, 1138, 1092, 1016, 820, 752,714 cm⁻¹; ¹H NMR (CDCl₃) δ 11.40 (br s, 1H), 9.34 (br s, 1H), 8.15-8.14(d, 1H), 7.84-7.80 (dd, 1H), 7.46-7.30 (m, 4H), 6.92-6.89 (d, 1H),4.10-4.07 (q, 2H), 3.13-3.08 (t, 2H); ¹³C NMR (CDCl₃) δ 179.2, 151.6,146.3, 141.3, 137.1, 130.2, 128.6, 113.5, 112.8, 46.8 and 34.2; MaldiTof found: 372.0 (M+1), calculated: 371.0; Anal. (C₁₄H₁₃BrClN₃S) C, H,N, S.

Chemical Synthesis III

Compounds III-1-3 were prepared as illustrated in Scheme 5. Thesynthesis involved condensing 2-amino-5-bromopyridine with1,1-thiocarbonyl diimidazole to furnish the required thiocarbonylderivative. Further reaction of this thiocarbonyl derivative with anappropirate amine gave 1-3 in good yields.

Physical Data of Synthesized Compounds:

N-[2-(1-piperidinylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-1)

Yield: 74%; mp 150-152°; ¹H NMR (CDCl₃) δ 11.53 (br s, 1H), 9.72 (br s,1H), 8.22 (d, 1H), 7.72-7.68 (dd, 1H), 6.95-6.92 (d, 1H), 3.84-3.78 (q,2H), 2.61-2.57 (t, 2H), 2.45 (br s, 4H), 1.64-1.48 (m, 6H); ¹³CNMR(CDCl₃) δ 178.1, 151.8, 146.3, 140.8, 113.5, 112.6, 56.1, 54.0, 43.0,26.3, and 24.3.

N-[2-(1-piperizinylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (1H-2)

Yield: 75%; mp 178-180° C.; ¹H NMR (CDCl₃) δ 11.50 (br s, 1H), 9.77 (brs, 1H), 8.19-8.18 (d, 1H), 7.75-7.71 (dd, 1H), 6.97-6.95 (d, 1H),3.87-3.86 (m, 2H), 3.63-3.60 (t, 2H), 3.45-3.42 (m, 3H), 2.74-2.69 (t,2H), 2.59-2.52 (m, 4H); ¹³C NMR(CDCl₃) δ 178.7, 151.8, 146.1, 141.0,113.7, 112.7, 55.2, 52.0, 51.9 and 45.8.

N-[2-(1-morpholinylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (III-3)

Yield: 65%; 124-126° C.; ¹H NMR (CDCl₃) δ 11.51 (br s, 1H), 9.23 (br s,1H), 8.25-8.24 (d, 1H), 7.75-7.71 (dd, 1H), 6.85-6.82 (d, 1H), 3.87-3.74(m, 6H), 2.68-2.54 (m, 6H); ¹³C NMR(CDCl₃) δ 178.5, 151.7, 146.4, 141.0,113.5, 112.7, 67.2, 55.4, 53.1, 42.5. Compound R Compound R I-1 pyridylII-1 piperidinyl I-3 piperidinyl III-2 piperozinyl I-4 2,5-dimethoxyphenyl III-3 morpholinyl II-1 o-methoxy phenyl II-6 m-chlorophenyl II-2o-fluorophenyl II-7 p-methoxy phenyl II-3 o-chlorophenyl II-8p-flurophenyl II-4 m-methoxy phenyl II-9 p-chlorophenyl II-5m-fluorophenyl

Example 5 Structure-Based Design and Docking of Novel PETT Derivativesinto Composite NNI Binding Pocket I

A novel model of the NNI binding pocket of RT was constructed bycarefully superimposing the coordinates of 9 individual RT-NNI crystalstructures and then generating a van der Waals surface which encompassedall of the overlaid ligands. The integrated structural information ofthis “composite binding pocket” revealed an unexpectedly different andmuch larger NNI binding site than shown in or predictable from any ofthe individual structures and served as a probe to more accuratelydefine the potentially usable space in the binding site (FIG. 2 a). Anumber of protein residues in contact with the inhibitors are relativelyflexible and vary from structure to structure. These residues includeTyr180, Tyr181, Tyr318, Try319, Phe227, Leu234, Trp229, Pro95, andGlu138 (from p51 subunit of RT). As shown in FIG. 2 b, the surface ofthe composite binding pocket is a short distance away from (<1.5 Å) oreven extends past RT residues 234-236, Y188, F227, and the backbone ofK101. This indicates that these residues are flexible and can bedisplaced by the right inhibitor. The composite binding pocket, unlikean individual crystal structure, is able to summarize the nature andextent of the flexibility of the active site residues. This allowedprediction of potential modification sites on the PETT derivatives Iafter positioning the compounds in the RT active site (see Methods).

A computer simulation of the binding of PETT compounds into the NNIbinding site of RT was accomplished using a molecular docking procedure.Docking of PETT and trovirdine into the NNI binding site required theuse of X-ray coordinates of an RT/NNI complex (in this case theRT/9-Cl-TIBO complex).

Upon binding to RT, the compound can fit into a butterfly-shaped NNIbinding site (described by Ding, J., et al., Nat. Struct. Biol., 1995,2, 407-415) (FIGS. 1B and 2). PETT and its derivatives such as compoundsI-1-4 could be viewed as two chemical groups linked together by athiourea group (Table 5). One half of the molecule is composed of a2-aminothiazole group (PETT) or a pyridylthiourea group (compoundsI-1-4) which forms an intramolecular hydrogen-bonded heterocyclic ring.The other half of the molecule is a phenyl or heterocyclic ringseparated from the thiocarbonyl group by an ethyl linker.

Once the final docked position of the molecule in the NNI site wasdetermined, the molecule was assigned a score, from which an estimationof the inhibition constant (K_(i) value) was determined (Table 5). Whentrovirdine was docked into the NNI binding site of RT it had a higherbinding score than PETT and fit into the butterfly-shaped binding regionwith one part residing in Wing 1 and the other in Wing 2 (FIG. 1B). Thering closest to the thiocarbonyl group resided near the Lys(K)101 loopand the other pyridyl ring was near Trp(W)229.

After docking and K_(i) estimation was completed for the PETTinhibitors, evaluation of the docked compounds in the active site of RTinvolved placing each compound into the composite binding pocket usingthe same orientation matrix utilized in its construction. Thepotentially flexible regions in the binding site were then readilyidentified as were atom sites for future derivatization of thecompounds. The area within Wing 2 and the residues near the thioureagroup seemed to be the most forgiving regions in the binding site of RT.This observation was also supported by the analysis of gaps inatom-to-atom contact between the protein and the inhibitor. TABLE 5Interaction scores, calculated K_(i) values, and measured IC₅₀ data forPETT derivatives I. PETT

I-1 to I-4

M.S.^(a) B.S.^(b) Lipo Ludi^(c) Ludi^(c) IC₅₀ R₁ R₂ (Å²) (%) Score ScoreK_(i)(μM) p24(μM) S.I.^(d) PETT phenyl 2-thiazolyl 254 84 625 562 2.4n.d. n.d. I-1 2-pyridyl 2-pyridyl 260 84 640 577 1.7   0.230 >435 I-22-pyridyl 2-(5-bromo) 276 84 679 616 0.7   0.007 >10⁴ Trovirdine pyridylI-3 1-piperidinyl 2-(5-bromo) 278 84 684 621 0.6 <0.001 >10⁵ pyridyl I-42,5- 2-(5-bromo) 317 84 779 716 0.2 <0.001 >10⁵ dimethoxy- pyridylphenyl AZT   0.008 6250^(a)MS, molecular surface area calculated using Connolly's MSprogram.(Connolly, M. L., Science, 1983, 221, 709-713) Defined asboundary of volume within any probe sphere (meant to represent a watermolecule) of given radius sharing no volume with hard sphere atoms whichmake up the molecule. Values are slightly smaller than thoseapproximated by Ludi program.^(b)BS, buried surface: percentage of molecular surface in contact withprotein calculated by Ludi based on docked positions. Based on publishedcrystal structures of RT complexes, our calculation shows that thesevalues could be as low as 77% (in RT-HEPT complex) and can be as high as90% (in RT-APA complex) but most of them including RT-MKC average around84%.^(c)Ludi Ki values were calculated based on the empirical score functionin Ludi program.(Bohm, H. J., J. Comput. Aided. Mol. Des., 1994, 8,243-256; 1996,) Ideal hydrogen bond distances and angles betweencompounds and protein are assumed in all cases for Ludi K_(i) and LudiScore calculation. In published crystal structures of RT complexes,hydrogen bond geometries are indeed close to ideal; the amide carbonylof residue A101 on a loop demonstrates substantial# flexibility which can accommodate the best geometry for hydrogenbonding. The number of rotatable bonds(=2) is used in the Ludicalculation to reflect the loss of binding energy due to freezing ofinternal degrees of freedom.^(d)Selectivity Index is equal to the ratio of fifty percent cytotoxicconcentration to IC₅₀.n.d., not determined.

Analysis of the molecular surface of the compounds in the NNI bindingsite of RT included visualization of spatial gaps between the compoundsand nearby residues of the RT protein, as described above for Example 1.The spheres generated are shown in FIG. 3, and indicate regions of thecompound which are probably not available for derivatization. FIG. 4shows the binding pocket embellished with a grid of red rods whichrepresent unoccupied space between the compound and active siteresidues, providing a complementary view to that shown by the spheres inFIG. 3. The grid illustrates the candidate sites for derivatization ofthe compound and, when used as a distance scale (the length of one rodrepresents 1 Å), also indicates the volume available for new functionalgroups. After the docked PETT compounds were subjected to the grid (gap)analysis, a number of gaps in the binding site were identified (FIGS.3-4), some of which could be filled by strategically designed functionalgroups on new PETT derivatives. It was postulated that a more efficientuse of such sterically allowed unoccupied spatial gaps in the bindingsite could be achieved by replacing the 2-pyridyl ring of trovirdinewith a 1-piperidinyl (compound I-3) or 2,5-dimethoxyphenyl moiety(compound 1-4) and yield potentially more active PETT compounds withlarger molecular surface areas, higher Ludi scores, and lower K_(i)values (Table 5).

Compounds I-1, I-3 and I-4 were subjected to the same docking procedureand K_(i) calculation used to analyze the parent compounds PETT andtrovirdine (compound 1-2). The molecular surface area of the compoundscalculated after docking increased in the following order: PETT,compound I-1, I-2 (trovirdine), 1-3, and 1-4. At docked positions, theatom surface area in contact with the protein residues constituted anaverage of 84% of the entire molecular surface (FIG. 3). We used thisaverage value in the calculation of the inhibitory constant (K_(i))based on the Ludi score function. Calculated K_(i) values for 1-3 and1-4 predicted that these compounds would have potency superior to thatof trovirdine. The calculated K_(i) values of our compound I-3 (0.6 μM),and compound I-4 (0.2 μM) were better than those of known compounds PETT(2.4 μM), compound 1-1 (1.7 μM) and trovirdine (0.7 μM).

Example 6 In Vitro Assays of Anti-HIV Activity Using PETT Derivatives I

The HIV-1 strain HTLV_(IIIB) (kindly provided by Dr. Neal T. Wetherall,VIROMED Laboratories, Inc.), was propagated in CCRF-CEM cells, and usedin in vitro assays of the anti-HIV_(IIIB)-HIV-1 activity of thesynthesized novel derivatives. Cell-free supernatants ofHTLV_(IIIB)-infected CCRF-CEM cells were harvested, dispensed into 1 mlaliquots, and frozen at −70° C. Periodic titration of stock virus wasperformed by examining its cytopathic effects in MT-2 cells followingthe procedures described in (Erice, et al., Antimicrob. Ag. Chemother.,1993, 37, 835).

Normal human peripheral blood mononuclear cells (PBMNC) fromHIV-negative donors were cultured 72 hours in RPMI 1640 supplementedwith 20% (v/v) heat-inactivated fetal bovine serum (FBS), 3%interleukin-2, 2 mM L-glutamine, 25 mM HEPES, 2 g/L NaHCO₃, 50 μg/mlgentamicin, and 4 μg/ml phytohemagglutinin prior to exposure to HIV-1.The incubated cells were then exposed to HIV-1 at a multiplicity ofinfection (MOI) of 0.1 during a one-hour adsorption period at 37° C. ina humidified 5% CO₂ atmosphere. Subsequently, infected cells werecultured in 96-well microtiter plates (100 μl/well; 2×10⁶ cells/ml) inthe presence of test compounds, including AZT as a control. Aliquots ofculture supernatants were removed from the wells on the 7th day afterinfection for p24 antigen assays. The methods used in the P24 assay wereas previously described in Uckun, et al., Antimicrobial Agents andChemotherapy, 1998, 42, 383; Zarling, et al., Nature, 1990, 347, 92-95;Erice, et al., Antimicrob. Ag. Chemother., 1993, 37, 835.

The applied p24 enzyme immunoassay (EIA) was the unmodified kineticassay commercially available from Coulter Corporation/Immunotech, Inc.(Westbrooke, Me.). In the assay, a murine monoclonal antibody againstHIV core protein is coated onto microwell strips. Antigen (HIV coreprotein) present in the test culture supernatant samples binds theantibody and the bound antibody-antigen complex is quantitated. Percentviral inhibition was calculated by comparing the p24 values from thetest substance-treated infected cells with p24 values from untreatedinfected cells (i.e., virus controls).

In addition, the activity of the test compounds to inhibit recombinantHIV-1 reverse transcriptase (rRT) activity was determined using theQuan-T-RT assay system (Amersham, Arlington Heights, Ill.), whichutilizes the scintillation proximity assay principle. The assay methodis described in Bosworth, N., et al., Nature, 1989, 341, 167-168. Datafor both bioassays is reported as IC₅₀ values.

In parallel with the bioactivity assays, the effects of the testcompounds on cell viability was also examined, using the MicrocultureTetrazolium Assay (MTA) described in Darling, et al., Nature, 1990, 347,92-95; Erice, et al., Antimicrob. Ag. Chemother., 1993, 37, 835. Inbrief, non-infected PBMNC were treated with test compounds or controlsfor 7 days under identical experimental conditions and2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazoliumhydroxide (XTT), was added to quantitative cellular proliferation.

An energy-minimized model of compound I-4 in the RT binding site had thelargest molecular surface area in contact with the protein and thusachieved the highest lipophilicity score. The docking studies indicatedthat the 2-methoxy group of compound I-4 is situated beneath the ethyllinker and fits favorably into a cavity of the binding pocket, providingcontact with protein residues that cannot be achieved by trovirdine.Likewise, the 5-methoxy group of compound I-4 provides close contactwith residues Pro95 and Trp229. The trend of the calculated K_(i) valuesaccurately predicted the trend of the experimentally determined IC₅₀values from HIV replication assays, as shown in Table 5, therebyproviding conclusive evidence of the practical utility of the compositemodel.

The lead compound, I-4 with the lowest calculated K_(i) values of theseries, was 8-times more potent than trovirdine against purifiedrecombinant HIV-RT using the cell-free Quan-T-RT system (IC50[rRT] was0.1 μM for I-4 versus 0.8 μM for trovirdine). Compound I-4 also elicitedpotent anti-HIV activity with IC₅₀ values of less than 0.001 μM in 3 of3 independent experiments which was consistently lower than the IC₅₀values for trovirdine (0.007 μM) and AZT (0.008 μM). None of the PETTderivatives were cytotoxic at concentrations as high as 100 μM.Therefore, the calculated selectivity index (IC₅₀[MTA]/IC₅₀[p24]) ofcompounds I-3 and I-4 were greater than 10⁵.

All active PETT compounds listed in Table 5 are able to form anintramolecular hydrogen bond between the nitrogen atom of pyridine orthiazole and an amide hydrogen of the thiourea group, as shown in Wing 1of FIG. 1B. The intramolecular hydrogen bond was also observed in oursmall molecule crystal structure of compound I-3 (data not shown). Theenergy gained by the formation of such a hydrogen bond has beenestimated to be about 5 kcal/mol (Bell, et al., J. Med. Chem., 1995, 38,4929-4936). Our docking results showed that the internal hydrogen bondkeeps the pyridyl thiourea (or thiazolylthiourea) in a more rigidconformation and allows the molecule to adopt the appropriate geometryto occupy Wing 1 of the binding site, and at the same time maintain ahydrogen bond with a backbone carbonyl of residue Lys 101 (FIG. 1B).

Compounds I-3 and I-4 differ from trovirdine at the proposed Wing 2binding region of the molecule. Compound I-3 has a heterocyclic ringwhich replaces the pyridyl ring and compound 4 has two methoxy groupsadded at meta and ortho positions of the phenyl ring. The molecularsurface areas of compounds I-3 and I-4 are larger than that oftrovirdine, as calculated from the coordinates of the predicted activeconformation obtained from docking. This larger surface area results ina better lipophilic score and lower calculated K_(i) value (Table 5).Both pyridylethyl and piperidinylethyl groups occupy the same region ofWing 2 near Tyr229 (FIGS. 2 and 5). Our composite binding pocket shows aspace large enough to accommodate a group larger than the pyridyl ringof trovirdine. Docking results and analyses of gaps indicate that thepyridyl ring of trovirdine has multiple sites which can be used forincorporation of larger groups. As shown in FIG. 5, there is sufficientspace surrounding the pyridylethyl ring for the addition of a two- tofour-atom substituent at any of the ring positions. Both sides of thepyridylethyl ring plane of trovirdine are relatively exposed in thepocket (FIG. 3A) and can accommodate additional substituents (FIG. 4A).This prediction was confirmed by the potency of compound I-4 (whichcontains ortho, meta-dimethoxy substituents), in inhibitng HIVreplication.

The piperidinyl group of I-3 is puckered and therefore occupies a largeroverall volume than the planar pyridyl ring of trovirdine and is inclose contact with residues Leu234 and Leu100, the latter of which canmutate to isoleucine, frequently found in a drug-resistant RT mutantstrain. In contrast to previously reported extensive attempts atexpanding within the pyridyl ring plane (Bell, et al., J. Med. Chem.,1995, 38, 4929-4936; Cantrell, A. S., et al., J. Med. Chem., 1996, 39,4261-4274; Ahgren, C., et al., Antimicrob. Agents Chemotherapy, 1995,39, 1329-1335), the success of our efforts at modification perpendicularto the ring plane introduces new possibilities to develop more potentinhibitors which combine both modifications. The piperidinyl ring isconformationally more flexible than an aromatic ring has the advantageof fitting an uncompromising binding pocket more effectively, despitethe expense paid for loss of entropy upon binding. The analysis shown inFIGS. 3, 4, and 5 provides new insights for modifications which aredifferent from those of trovirdine derivatives. Various combinations ofdouble substitutions at axial or equatorial positions of the piperidinylring generate derivatives with a broader range of curvatures thantrovirdine derivatives and better fit Wing 2 which itself contains somecurvature.

In summary, a composite binding pocket was constructed which integratedall available crystal structure information about the NNI binding siteof RT. This novel computer-generated model was an unexpectedly effectivetool that helped to much better comprehend the flexible nature of thebinding pocket and to identify specific areas for structuralimprovements of the inhibitors. Nine lead NNI compounds from publishedcrystal structures were analyzed. With all strategies combined, a numberof previously unknown candidate sites for developing more potentderivatives of PETT were identified, such as substituting a bulkierpiperidinyl group or an ortho/meta substituted phenyl group in place ofan unsubstituted ring which resulted in enhanced inhibitory activity.The presented experimental results demonstrate that two novel PETTderivatives which resulted from our structure-based design efforts usingthe composite binding pocket are remarkably potent and noncytotoxicanti-HIV agents with unprecedented selectivity indices of >10⁵. Thesuperior activity of these designed PETT compounds would not have beenpredictable from existing information about trovirdine alone, or fromany single crystal structure of RT complexed with an NNI.

Example 7 Structure-Based Design and Docking of PETT Derivatives intoComposite NNI Binding Pocket II

The PETT derivatives II, synthesized as described above for Example 4,were analyzed for fit into the NNI binding pocket. Target compounds werealso analyzed for anti-viral activity in p24 enzyme immunoassays andalso for the ability to inhibit HIV reverse transcriptase activity,using rRT. Methods for these biological assays are described above forExample 6.

A computer simulation of the binding of the target PETT derivatives intothe NNI binding site of RT was accomplished using a molecular dockingprocedure. Docking of the compounds into the NNI binding site requiredthe use of X-ray coordinates of an RT/NNI complex (in this case theRT/9-Cl-TIBO complex).

Trovirdine derivatives could be viewed as two chemical groups linkedtogether by a thiourea group (Table 6). One half of the molecule iscomposed of a pyridylthiourea group (compounds II-1-9) which forms anintramolecular hydrogen-bonded heterocyclic ring (shown in trovirdinestructure). The other half of the molecule is a pyridyl ring separatedfrom the thiocarbonyl group by an ethyl linker.

When trovirdine was docked into the NNI binding site of RT, it fit intothe butterfly-shaped binding region (described by Ding, et al., Nat.Struct. Biol., 1995, 2, 407-415) with one part of the molecule residingin Wing 1 and the other in Wing 2. The ring closest to the thiocarbonylgroup resided near the Lys(K)101 loop and the other pyridyl ring wasnear Trp(W)229.

Compounds II-1-9 were positioned according to this binding mode into theRT/9-Cl-TIBO active site by a docking procedure described above forExample 1. The results are shown in FIG. 6. One of the NH groups of thethiourea part of these compounds consistently formed a hydrogen bondwith the backbone of K101.

Once the final, energetically favored docked position of the molecule inthe NNI site was determined, a LUDI score was assigned, from which anestimation of the inhibition constant (K value) was determined (Table6). The calculated K_(i) values, ranging from 0.4 μM to 0.8 μM suggestedthat compounds II-2-7 would be active inhibitors of RT. The modelingdata, shown below in Table 6, predicted that compounds II-2 to 1′-7would be as potent as or more potent than trovirdine for inhibiting RT.The data for the bioassay of RT inhibition follows this prediction.TABLE 6 Interaction scores, K_(i) values, and measured IC₅₀ data for aseries of PETT derivatives. II-1 to II-9

MS^(a) BS^(b) LIPO K_(i)(calc) IC₅₀ rRT* IC₅₀ p24 Compound X (Å²) (%)Score (μM)^(c) (μM) (μM) SI^(d) II-1 o-OMe 282 82% 678 1.2 1.0 0.01  >1× 10⁴ II-2 o-F 281 82% 674 0.8 0.6 <0.001   >1 × 10⁵ II-3 o-Cl 285 83%694 0.5 0.7 <0.001   >1 × 10⁵ II-4 m-OMe 296 84% 729 0.4 0.4 0.003 >3 ×10⁴ II-5 m-F 282 83% 687 0.6 0.7 <0.001   >1 × 10⁵ II-6 m-Cl 283 81% 6720.8 3.1 N.D. N.D. II-7 p-OMe 302 83% 734 0.6 0.9 0.015 >6 × 10³ II-8 p-F284 81% 674 7.8 6.4 N.D. N.D. II-9 p-Cl 293 81% 696 4.7 2.5 N.D. N.D.trovirdine N.A. 276 84% 679 0.7 0.8 0.007 >1 × 10⁴ AZT N.A. N.A. N.A.N.A. N.A. >100 0.004   7 × 10³*rRT, recombinant HIV reverse transcriptase assay^(a)MS, molecular surface area calculated using Connolly's MSprogram.(Connolly, Science, 1983, 221, 709-713) Defined as boundary ofvolume within any probe sphere (meant to represent a water molecule) ofgiven radius sharing no volume with hard sphere atoms which make up themolecule. Values are slightly smaller than those approximated by Ludiprogram.^(b)BS, buried surface: percentage of molecular surface in contact withprotein calculated by Ludi based on docked positions. Based on publishedcrystal structures of RT complexes, our calculation shows that thesevalues could be as low as 77% (in RT-HEPT complex) and can be as high as90% (in RT-APA complex) but most of them average around 84%.^(c)Ludi K_(i) values were calculated based on modified empirical scorefunction in the Ludi program as described for Example 1. (Bohm, J.Comput. Aided. Mol. Des., 1994, 8, 243-256; 1996,) Ideal hydrogen bonddistances and angles between compounds and protein are assumed in allcases for Ludi Score and K_(i) calculation. In published crystalstructures of RT complexes, hydrogen bond geometries are indeed close toideal; the amide carbonyl of residue A101# on a loop demonstrates substantial flexibility which can accommodatethe best geometry for hydrogen bonding. The number of rotatable bonds(2, or 2 + n for n methoxy groups) is used in the Ludi calculation toreflect loss of binding energy due to freezing of internal degrees offreedom.^(d)SI (selectivity index) = IC₅₀[MTA]/IC₅₀[p24]). IC₅₀[MTA] valueswere >100 μM for compounds II-1-9, as well as trovirdine. IC₅₀[MTA] forAZT was 50 μM.N.D., not determined, for compounds with IC₅₀[rRT] greater than 1.0 μM.N.A., not applicable.

Example 8 In Vitro Assays of PETT Derivatives II

Methoxy Substitutions

The estimated K_(i) values accurately predicted the trend of themeasured IC₅₀[rRT] values for the inhibition of recombinant HIV RT.Compound II-4 had the lowest K_(i) value. The docking results showedthat the meta-methoxy group of II-4 is situated near Pro95 and Trp229 inthe binding site, providing contact with these protein residues whichcannot be achieved by trovirdine (FIG. 5). Based on the IC₅₀ [rRT]values of all methoxy compounds, the meta-methoxy substituted compound1′-4, which had a K_(i) value of 0.4 μM, showed greater inhibitoryactivity against recombinant HIV RT and it was approximately 2-fold morepotent than trovirdine (IC₅₀[rRT] was 0.4 μM for compound II-4 versus0.811M for trovirdine). Compound II-4 abrogated HIV replication in humanperipheral blood mononuclear cells at nanomolar concentrations with anIC₅₀ value of 3 nM and a selectivity index (SI) of >3×10⁴ (Table 6).

Fluorine Substitutions

Among the fluorine (F) substituted compounds II-2, II-5, and II-8, bothmeta and ortho fluoro compounds were at least 7-fold more active thantrovirdine (IC₅₀[p24]<1 nM) (Table 6). Based on the IC₅₀[rRT] values,compounds with F substitutions at the meta and ortho positions hadnearly the same inhibitory activity against recombinant HIV RT but thepara-F substituted compound was 10-fold less active. The color-codedcomposite binding pocket (FIG. 5) also shows that Wing 2 is mostlyhydrophobic except for the region near the ortho positions on both sidesof the phenyl ring where polar groups such as halogen atoms would becompatible. Trovirdine, however, lacks such ring substitutents whichcould provide favorable interactions with these regions of the bindingsite based on our modeling. Substitutions at the meta position could beon the polar region or the hydrophobic region depending on the chemicalgroup and its consequent conformational change (FIG. 5). The m-Fsubstituent of compound II-5 is probably exposed to the polar (blue)region and therefore is as active as the o-F group which would also beexposed to the polar region according to our modeling. The trend inIC₅₀[rRT] values observed for F-substituted compounds may reflect such apreference. The p-F atom, which is small in size but electronegative,may not be compatible with the location of the ring plane of nearbyhydrophobic Trp229 and could contribute to the lower activity. Wepostulate that this same incompatibility should be observed for anyother highly hydrophilic group at the para position, and that anadditional binding penalty be imposed to better quantitate such featureswhen undertaking modeling studies.

Chlorine Substitutions

Chlorine (Cl) substituted compounds II-3, II-6, and II-9 show a trend ofobserved biological activities which differs from that of both thefluorine and methoxy compounds. Like the p-F substituted compound whichwas less active than other F-substituted compounds, the p-Cl compoundwas less active than the o-Cl compound based on the IC₅₀[rRT] values.Unlike the m-F substituted compound which was as active as the o-Fsubstituted compound, the m-Cl compound was not as active as the o-Clsubstituted compound. According to our modeling, o-Cl is the most likelysubstituent to be situated near a limited polar region at Wing 2, aninteraction which would be favorable. The o-Cl compound, like the o-Fcompound discussed above, was in fact more active than trovirdine, aswas predicted by the modeling procedure and by the use of the compositebinding pocket.

Hydrophobic Group Preferred at the Para Position

When IC₅₀[rRT]values of all compounds with para substitutions arecompared (II-7-9), a distinct trend is evident: the p-methoxy (OMe)compound (7) is favored over the p-halogen group compounds (II-8 andII-9) (Table 6). Only the p-OMe substituted PETT derivative, compoundII-7, is comparable to trovirdine in its inhibitory activity againstrecombinant HIV RT. Compound II-7 inhibited HIV replication inperipheral blood mononuclear cells with an IC₅₀ value of 15 nM (Table6). This p-OMe preference is consistent with the understanding of thecolor-coded composite binding pocket at Wing 2, where the binding pocketresidues near the para position are relatively hydrophobic. One canreasonably assume, based on chemical intuition and the availableinhibition data which is consistent with the modeling, that parasubstituted hydrophobic groups positioned near a hydrophobic region ofthe pocket are most preferred, followed by halogens, and finallyhydrophilic groups.

Conclusions

In summary, the data revealed the following structure-activityrelationships affecting the potency of PETT derivatives withsubstitutions on various positions of the phenyl ring:

-   1) methoxy substitution is more favorable at the meta position than    at the ortho or para positions;-   2) fluorine substitution is favorable at ortho and meta positions    but not at the para position;-   3) chlorine substitution is favorable only at the ortho position;-   4) a hydrophobic group is more desirable than a polar group or    hydrophilic group at the para position. These results were generally    consistent with predictions made during modeling.

The use of the composite NNI binding pocket allowed the identificationand structure-based design of at least 3 promising PETT derivatives withortho-F (1′-2), ortho-Cl (II-3), and meta-F (II-5) substituents on thephenyl ring. These novel PETT derivatives were more active thantrovirdine (as predicted) or AZT and showed potent anti-HIV activitywith IC₅₀ [p24] values<1 nM and selectivity indices (SI) of >100,000(Table 6).

Example 9 Design of Heterocyclic PETT Derivatives III

In the course of the search for potent NNIs, a computer model has beendeveloped in which a composite binding pocket was constructed from nineindividual crystal structures of RT-NNI complexes. Modeling studies leadto the identification of a number of NNIs with IC₅₀ values beyond 1 nMfor the inhibition of HIV replication (measured by p24 production inHIV-infected human peripheral blood mononuclear cells) and showed nodetectable cytotoxicity against human T-lymphocytes (inhibition ofcellular proliferation was >100 μM as measured by MTA).

The detailed analysis of trovirdine, a potent PETT derivative, revealedmultiple sites which can be used for the incorporation of largerfunctional groups. In the composite binding pocket, the dockedtrovirdine molecule showed a lot of usable space surrounding the pyridylring, the ethyl linker and near the 5-bromo position (shown in structureof PETT derivative). It was proposed that efficient use of this space bystrategically designed functional groups would lead to high affinitybinding and ultimately result in better inhibitors.

III 1 2 3 X = CH₂ NH O

The effect of systematic substitutions of the phenyl ring of trovirdinewith various heterocyclic rings was studied. This provides analternative strategy to fit the compound into the relatively flexibleand spacious Wing 2 region (as illustrated by the composite bindingpocket). In the subsequent modeling studies these heterocyclic rings,which have a larger volume than the pyridyl ring of trovirdine, wereshown to better fill the Wing 2 region of the composite binding pocket.

The piperidinyl, piperzinyl and morpholinyl rings of compounds III-1-3are puckered and therefore occupy a larger overall volume than theplanar pyridyl ring of trovirdine and are in close contact with residuesLeu234 and Leu100, the latter of which can mutate to isoleucine,frequently found in a drug-resistant RT mutant strain. The encouragingresults from efforts to make modifications perpendicular to the ringplane introduces new possibilities to develop more potent inhibitors ofRT.

The heterocyclic rings which are conformationally more flexible than anaromatic ring may have the advantage of fitting an uncompromisingbinding pocket more effectively, despite the expense paid for loss ofentropy upon binding. Various combinations of double substitutions ataxial or equatorial positions of these heterocyclic rings would generatederivatives with a broader range of curvatures than trovirdinederivatives and would serve to better fit Wing 2 which itself containssome curvature.

Example 10 In Vitro Assays of Anti-HIV-1 Activity Using PETT DerivativesIII

Compounds III-1 to III-3 were tested for anti-HIV activity inHTLV_(IIIB)-infected peripheral blood mononuclear cells. Anti-HIVactivity was tested using the p24 immunoassay described above forExample 6. Cytotoxicity was also analyzed using a Microculturetetrazolium Assay (MTA), as described above for Example 6.

The data below in Table 7 show the inhibitory effects of PETTderivatives (compounds III-1-3) on p24 production in HIV-infectedperipheral blood mononuclear cells and on viability of peripheral bloodmononuclear cells. IC₅₀ values represent the concentration required toinhibit by 50% the activity of HIV replication as measured by assays ofp24 production (IC₅₀ [p24]) or the concentration required to decreasecellular proliferation by 50% as measured by MTA (IC₅₀ [MTA]) (Uckun, etal., Antimicrobial Agents and Chemotherapy, 1998, 42, 383; Zarling, etal., Nature, 1990, 347, 92-95; Erice, et al., Antimicrob. Ag.Chemother., 1993, 37, 835).

All three compounds III-1-3 were more potent than trovirdine forinhibitition of HIV. Our lead heterocyclic PETT derivatives,N-[2-(1-piperidinylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (compoundIII-1) and N-[2-(1-morpholinylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea(compound 3) elicited potent anti-FIV activity with IC₅₀ values lessthan 1 nM for the inhibition of HIV replication (measured by p24production in HIV-infected human peripheral blood mononuclear cells) andshowed no detectable cytotoxicity (inhibition of cellular proliferationwas >100 μM as measured by MTA) (Table 7). TABLE 7

IC₅₀[p24] IC₅₀[MTA] Compound R (μM) (μM) SI III-1 piperdinyl<0.001 >100 >1 × 10⁵ III-2 piperazinyl 0.002 >100 >5 × 10⁴ III-3morpholinyl <0.001 >100 >1 × 10⁵ Trovirdine pyridyl 0.007 >100 >1 × 10⁴AZT — 0.004 50   7 × 10³

Example 11 “SeeGap” Program for Analysis of Gap Space

To analyze the gap space between the binding pocket and complexed NNI,the “SeeGap” program was developed. The following instructions are foruse of the program, whose code is listed below in Table 8:

Preparation:

-   1. Extract the source codes at the lines indicated. The first    program is a C-shell command file and should be named as “SeeGap”;    the second program should be named as “pdbmax.f”; the third    “gridbox.f” and fourth “chgcolor.f”.-   2. Compile the source codes: for the first, chmod+x SeeGap; the    second, third, and fourth by “f77-o file file.f”.-   3. You should now have the executive versions of the programs named    as “SeeGap”, “pdbmax”, “gridbox” and “chgcolor”. The preparation is    ready.    Use the program:-   1. Open “insightII” window, and read in the coordinates of the    protein and the coordinates of the ligand. Next, assign the    potential to both coordinates by builder module within “insightII”    (see insight II manual).-   2. Position the ligand in the binding site by a docking procedure,    if the position of the ligand is unknown.-   3. Using subset/interface command, determine the coordinates of the    protein that immediately surround the ligand by a defined distance,    e.g., 7 angstroms. Write out the coordinates and name it as    “bind.pdb”; write out the coordinates of the ligand and name it as    “ligand.pdb”.-   4. Adjust the input parameters in the command file “SeeGap” as    appropriate.-   5. Run the program by typing “SeeGap ligand.pdb bind.pdb>out&”.-   6. The results should be in three files: contact.pdb, which    represents the grid points on the surface of the ligand and in    contact with the protein residues; gap.pdb, which represents the    grid points available for modification; and lig.pdb, which    represents the grid points covering the ligand.

7. Use a molecular graphics software to display these coordinates. TABLE8A C-shell command file ″SeeGap″ #######C-shell command file “SeeGap”,## cut below #!/bin/csh # chen mao, Nov. 8, 1997 grep ″ATOM ″ $1 >fort.1grep ″ATOM ″ $2 >fort.2 # modify expansion value (5.0 A) for the ligand/usr2/mao/local/bin/pdbmax <<eof 5.0 eof # modify the grid (1.0 A),too-small-grids may waste time /usr2/mao/local/bin/gridbox <<eof 1.0 eof# modify the distance cutoff considered to be close/usr2/mao/local/bin/chgcolor <<eof 2.0 eof grep ″  H ″fort.30>contact.pdb grep ″END ″ fort.30>>contact.pdb grep ″  N ″fort.20>lig.pdb grep ″END ″ fort.30>>lig.pdb grep ″ OH2″ fort.30>gap.pdbgrep ″END ″ fort.30>>gap.pdb /bin/rm fort.1 fort.2 fort.30 fort.20

TABLE 8B Program ″pdbmax.f″ to Determine Boundaries ## PROGRAM“pdbmax.f” TO DETERMINED THE BOUNDARY OF ### THE COORDINATES, cut below# xmin=9999.0 xmax=−9999.0 ymin=9999.0 ymax=−9999.0 zmin=9999.0zmax=−9999.0 open(unit=99, file=″boundary.out″,status=″unknown″) read(*,*)add 20 read(1,′(30x,3f8.3)′,end=999)x,y,z if (x.lt.xmin) xmin=x if(y.lt.ymin) ymin=y if (z.lt.zmin) zmin=z if (x.gt.xmax) xmax=x if(y.gt.ymax) ymax=y if (z.gt.zmax) zmax=z go to 20 1000format(a4,i7,2x,a1,a2,1x,a3,2x,i4,4x,3f8.3,2f6.2) 999 continuewrite(*,′(″the extreme of the coordinates are″)′)write(*,′(6(3x,f6.1))′)xmin,xmax,ymin,ymax,zmin,zmax xmin=xmin−addymin=ymin−add zmin=zmin−add xmax=xmax+add ymax=ymax+add zmax=zmax+addwrite(99,′(6(3x,f6.1))′)xmin,xmax,ymin,ymax,zmin,zmax stop end

TABLE 8C Program ″gridbox.f″ to Generate Grids ####### PROGRAM“gridbox.f” TO GENERATE GRIDS FOR THE #BINDING SITE, cut belowCHARACTER*1 ATOM1 character*2 ATOM2 CHARACTER*4 CHN character*4 RESinteger xs, ys, zs parameter q=1.0, w=0.0 write(*,′(″step size in A″)′)open(unit=99,file=″boundary.out″,status=″old″,readonly) read(*,*)stepCHN=‘ATOM’ RES=′TIP3′ ATOM1=‘O’ ATOM2=′H2′ ICNTS=0 C read the boundaryof the box to generate grid write(*,′(″six min max values ″)′) read(99,*)xmin, xmax, ymin, ymax, zmin, zmax s=(xmax−xmin)/step xs=ss=(ymax−ymin)/step ys=s s=(zmax−zmin)/step zs=s if(xs.lt.0.0.or.ys.lt.0.0.or.zs.lt.0.0) then write(*,′(″nonsense input″)′)go to 999 end if write(*,*)xs,ys,zs inum=xs*ys*zs write(*,*)inum if(inum.gt.25000) then write(*,′(″too many grids″)′) go to 999 end if do100 n=1, zs do 100 m=1, ys do 100 l=1, xs x1=xmin+float(l)*stepy1=ymin+float(m)*step z1=zmin+float(n)*step icnts=icnts+1 100 write(10,1000) CHN,ICNTS,ATOM1,ATOM2,RES, 1 icnts, x1,y1,z1,Q,W 1000format(A4,I7,2X,A1,A2,1X,A4,I6,3X,3F8.3,2F6.2) C write (10,′(″END ″)′)999 stop end

TABLE 8D Program ″chgcolor.f″ to Determine Contact Area and GAP #####PROGRAM “chgcolor.f” TO DETERMINE THE CONTACT ## AREA AND GAP, cut below# character*1 atom1, zatom1 character*2 atom2, zatom2 CHARACTER*4 chn,zchn character*4 res, zres integer iatom, izatom, ires, izres real u, v,w, q, zq, windex, zw C set for delta distance value, please revise Cparameter da=1.5 write(*,′(″distance cutoff″)′) C dal is for hydrogen,da2 for other kinds read (*,*)da C read (*,*)da1, da2 100 read (10,1000,end=199) chn,iatom,atom1,atom2,res, 1 ires, u,v,w,q,windex rewind 1 130read(1, 1000, end=198) zchn,izatom,zatom1,zatom2,zres, 1izres,x,y,z,zq,zw C if (zatom1.eq.″H″) then C da=da1 C go to 133 C endif C da=da2 133 delx=abs(u−x) dely=abs(v−y) delz=abs(w−z)if(delx.lt.da.and.dely.lt.da.and.delz.lt.da ) thendist=sqrt(delx*delx+dely*dely+delz*delz) if(dist.lt.da) thenwindex=windex+1.0 atom1=″N″ atom2=″ ″ go to 198 end if end if go to 130198 write (20,1000)chn,iatom,atom1,atom2,res, 1 ires, u,v,w,q,windex goto 100 199 continue rewind 20 200 read (20,1000, end=299)chn,iatom,atom1,atom2,res, 1 ires, u,v,w,q,windex rewind 2 230 read(2,1000, end=298) zchn,izatom,zatom1,zatom2,zres, 1 izres,x,y,z,zq,zw C if(zatom1.eq.″H″) then C da=da1 C go to 233 C end if C da=da2 233delx=abs(u−x) dely=abs(v−y) delz=abs(w−z)if(delx.lt.da.and.dely.lt.da.and.delz.lt.da ) thendist=sqrt(delx*delx+dely*dely+delz*delz) if(dist.lt.da) thenwindex=windex+1.0 atom1=″C″ atom2=″ ″ go to 298 end if end if go to 230298 continue if (windex.eq.2.0) then atom1=″H″ atom2=″ ″ end ifwrite (30,1000)chn,iatom,atom1,atom2,res, 1 ires, u,v,w,q,windex go to200 299 continue write(30,′(″END ″)′) stop 1000format(A4,I7,2X,A1,A2,1X,A4,1x,I5,3X,3F8.3,2F6.2) end######################################

TABLE 9 Coordinates of Composite Binding Pocket These coordinates can beentered into a molecular graphics program to generate a molecularsurface representation of the composite binding pocket, which then canbe used to design and evaluate inhibitors of RT. ATOM 1 O H2O 1 144.048−24.778 68.464 inf inf ATOM 2 O H2O 2 144.416 −24.592 68.433 inf infATOM 3 O H2O 3 144.416 −24.225 68.423 inf inf ATOM 4 O H2O 4 143.694−25.486 68.876 inf inf ATOM 5 O H2O 5 144.048 −25.306 68.683 inf infATOM 6 O H2O 6 144.749 −25.257 68.756 inf ATOM 7 O H2O 7 143.349 −24.94468.703 inf inf ATOM 8 O H2O 8 144.790 −24.969 68.630 inf inf ATOM 9 OH2O 9 143.080 −24.603 68.775 inf inf ATOM 10 O H2O 10 145.130 −24.58168.682 inf inf ATOM 11 O H2O 11 143.639 −24.225 68.487 inf inf ATOM 12 OH2O 12 145.513 −24.404 68.846 inf inf ATOM 13 O H2O 13 143.655 −23.83268.549 inf inf ATOM 14 O H2O 14 145.157 −23.856 68.637 inf inf ATOM 15 OH2O 15 143.471 −23.455 68.774 inf inf ATOM 16 O H2O 16 144.786 −23.48068.619 inf inf ATOM 17 O H2O 17 143.670 −23.285 68.803 inf inf ATOM 18 OH2O 18 144.785 −23.149 68.737 inf inf ATOM 19 O H2O 19 144.417 −22.94968.853 inf inf ATOM 20 O H2O 20 143.693 −25.667 69.048 inf inf ATOM 21 OH2O 21 144.417 −25.702 69.012 inf inf ATOM 22 O H2O 22 143.280 −25.55469.161 inf inf ATOM 23 O H2O 23 145.154 −25.515 69.200 inf inf ATOM 24 OH2O 24 142.936 −24.965 69.009 inf inf ATOM 25 O H2O 25 142.683 −24.61869.149 inf inf ATOM 26 O H2O 26 142.673 −24.225 69.139 inf inf ATOM 27 OH2O 27 146.037 −24.225 69.239 inf inf ATOM 28 O H2O 28 146.042 −23.85669.233 inf inf ATOM 29 O H2O 29 145.586 −23.456 68.921 inf inf ATOM 30 OH2O 30 143.152 −23.144 69.225 inf inf ATOM 31 O H2O 31 145.515 −23.12569.025 inf inf ATOM 32 O H2O 32 143.661 −22.890 69.155 inf inf ATOM 33 OH2O 33 144.786 −22.742 69.007 inf inf ATOM 34 O H2O 34 144.063 −22.60269.236 inf inf ATOM 35 O H2O 35 144.048 −26.097 69.620 inf inf ATOM 36 OH2O 36 144.417 −25.997 69.413 inf inf ATOM 37 O H2O 37 143.287 −25.73069.365 inf inf ATOM 38 O H2O 38 145.148 −25.868 69.584 inf inf ATOM 39 OH2O 39 142.892 −25.364 69.350 inf inf ATOM 40 O H2O 40 142.606 −25.13069.584 inf inf ATOM 41 O H2O 41 145.857 −25.125 69.596 inf inf ATOM 42 OH2O 42 145.964 −24.629 69.323 inf inf ATOM 43 O H2O 43 146.208 −24.25869.503 inf inf ATOM 44 O H2O 44 142.554 −23.662 69.558 inf inf ATOM 45 OH2O 45 142.828 −23.175 69.610 inf inf ATOM 46 O H2O 46 143.260 −22.85869.517 inf inf ATOM 47 O H2O 47 145.718 −22.739 69.559 inf inf ATOM 48 OH2O 48 143.886 −22.425 69.590 inf inf ATOM 49 O H2O 49 144.975 −22.34569.548 inf inf ATOM 50 O H2O 50 144.786 −22.277 69.595 inf inf ATOM 51 OH2O 51 144.048 −26.251 69.938 inf inf ATOM 52 O H2O 52 144.994 −26.12569.920 inf inf ATOM 53 O H2O 53 145.525 −25.701 69.751 inf inf ATOM 54 OH2O 54 142.858 −25.603 69.941 inf inf ATOM 55 O H2O 55 142.410 −24.95669.939 inf inf ATOM 56 O H2O 56 146.247 −24.586 69.759 inf inf ATOM 57 OH2O 57 146.322 −24.242 69.726 inf inf ATOM 58 O H2O 58 146.447 −23.85669.936 inf inf ATOM 59 O H2O 59 146.368 −23.509 69.971 inf inf ATOM 60 OH2O 60 146.277 −23.296 69.932 inf inf ATOM 61 O H2O 61 145.876 −22.76269.762 inf inf ATOM 62 O H2O 62 143.833 −22.310 69.916 inf inf ATOM 63 OH2O 63 145.829 −22.628 69.962 inf inf ATOM 64 O H2O 64 145.143 −22.23069.948 inf inf ATOM 65 O H2O 65 144.048 −26.591 70.339 inf inf ATOM 66 OH2O 66 144.605 −26.461 70.287 inf inf ATOM 67 O H2O 67 144.849 −26.35070.242 inf inf ATOM 68 O H2O 68 143.010 −25.838 70.326 inf inf ATOM 69 OH2O 69 145.844 −25.653 70.169 inf inf ATOM 70 O H2O 70 142.505 −25.25370.305 inf inf ATOM 71 O H2O 71 146.408 −25.313 70.366 inf inf ATOM 72 OH2O 72 142.287 −24.619 70.305 inf inf ATOM 73 O H2O 73 142.270 −24.22570.305 inf inf ATOM 74 O H2O 74 146.581 −23.856 70.155 inf inf ATOM 75 OH2O 75 146.640 −23.667 70.298 inf inf ATOM 76 O H2O 76 146.387 −23.16570.341 inf inf ATOM 77 O H2O 77 146.235 −22.946 70.319 inf inf ATOM 78 OH2O 78 145.533 −22.364 70.118 inf inf ATOM 79 O H2O 79 144.038 −22.15670.305 inf inf ATOM 80 O H2O 80 145.471 −22.274 70.333 inf inf ATOM 81 OH2O 81 144.048 −27.016 70.623 inf inf ATOM 82 O H2O 82 144.634 −26.84170.626 inf inf ATOM 83 O H2O 83 144.819 −26.507 70.435 inf inf ATOM 84 OH2O 84 145.332 −26.427 70.685 inf inf ATOM 85 O H2O 85 145.880 −26.22870.717 inf inf ATOM 86 O H2O 86 142.907 −25.909 70.653 inf inf ATOM 87 OH2O 87 146.588 −25.657 70.623 inf inf ATOM 88 O H2O 88 147.374 −25.70070.660 inf inf ATOM 89 O H2O 89 148.108 −25.686 70.594 inf inf ATOM 90 OH2O 90 142.531 −25.283 70.673 inf inf ATOM 91 O H2O 91 147.001 −25.53070.644 inf inf ATOM 92 O H2O 92 148.427 −25.333 70.643 inf inf ATOM 93 OH2O 93 146.982 −24.943 70.558 inf inf ATOM 94 O H2O 94 148.109 −25.14070.625 inf inf ATOM 95 O H2O 95 147.195 −24.587 70.651 inf inf ATOM 96 OH2O 96 147.177 −24.225 70.696 inf inf ATOM 97 O H2O 97 142.471 −23.51570.647 inf inf ATOM 98 O H2O 98 142.595 −23.318 70.666 inf inf ATOM 99 OH2O 99 142.934 −22.926 70.677 inf inf ATOM 100 O H2O 100 146.583 −22.96970.735 inf inf ATOM 101 O H2O 101 146.022 −22.436 70.730 inf inf ATOM102 O H2O 102 144.417 −22.087 70.674 inf inf ATOM 103 O H2O 103 145.844−22.277 70.742 inf inf ATOM 104 O H2O 104 144.233 −27.553 71.039 inf infATOM 105 O H2O 105 143.655 −27.432 70.974 inf inf ATOM 106 O H2O 106144.442 −27.438 70.968 inf inf ATOM 107 O H2O 107 142.971 −26.975 71.068inf inf ATOM 108 O H2O 108 144.850 −26.872 70.763 inf inf ATOM 109 O H2O109 142.790 −26.440 71.066 inf inf ATOM 110 O H2O 110 145.888 −26.61471.059 inf inf ATOM 111 O H2O 111 147.185 −26.441 71.041 inf inf ATOM112 O H2O 112 148.109 −26.648 71.020 inf inf ATOM 113 O H2O 113 148.669−26.449 71.032 inf inf ATOM 114 O H2O 114 146.285 −26.324 70.974 inf infATOM 115 O H2O 115 147.001 −26.084 70.828 inf inf ATOM 116 O H2O 116148.503 −26.108 70.772 inf inf ATOM 117 O H2O 117 142.649 −25.772 70.972inf inf ATOM 118 O H2O 118 142.535 −25.326 71.039 inf inf ATOM 119 O H2O119 142.463 −24.937 71.041 inf inf ATOM 120 O H2O 120 148.837 −24.97370.888 inf inf ATOM 121 O H2O 121 147.762 −24.573 70.772 inf inf ATOM122 O H2O 122 149.033 −24.594 71.039 inf inf ATOM 123 O H2O 123 148.108−24.225 70.852 inf inf ATOM 124 O H2O 124 142.459 −23.880 71.019 inf infATOM 125 O H2O 125 148.477 −23.866 70.928 inf inf ATOM 126 O H2O 126142.550 −23.661 71.054 inf inf ATOM 127 O H2O 127 147.710 −23.518 70.952inf inf ATOM 128 O H2O 128 148.845 −23.672 71.048 inf inf ATOM 129 O H2O129 147.390 −23.272 70.974 inf inf ATOM 130 O H2O 130 143.004 −22.99671.018 inf inf ATOM 131 O H2O 131 147.021 −22.918 71.009 inf inf ATOM132 O H2O 132 143.843 −22.331 71.057 inf inf ATOM 133 O H2O 133 144.057−22.209 71.039 inf inf ATOM 134 O H2O 134 145.155 −22.003 70.856 inf infATOM 135 O H2O 135 146.253 −22.218 71.067 inf inf ATOM 136 O H2O 136145.894 −21.890 71.108 inf inf ATOM 137 O H2O 137 143.673 −27.752 71.404inf inf ATOM 138 O H2O 138 144.425 −27.759 71.401 inf inf ATOM 139 O H2O139 142.960 −27.339 71.427 inf inf ATOM 140 O H2O 140 145.148 −27.35371.418 inf inf ATOM 141 O H2O 141 145.550 −27.062 71.366 inf inf ATOM142 O H2O 142 146.233 −26.749 71.320 inf inf ATOM 143 O H2O 143 147.403−26.893 71.480 inf inf ATOM 144 O H2O 144 147.735 −26.822 71.219 inf infATOM 145 O H2O 145 148.468 −26.781 71.247 inf inf ATOM 146 O H2O 146142.643 −26.440 71.276 inf inf ATOM 147 O H2O 147 147.003 −26.730 71.337inf inf ATOM 148 O H2O 148 142.446 −26.051 71.452 inf inf ATOM 149 O H2O149 149.369 −26.060 71.434 inf inf ATOM 150 O H2O 150 149.447 −25.71971.367 inf inf ATOM 151 O H2O 151 142.424 −24.951 71.422 inf inf ATOM152 O H2O 152 149.685 −24.933 71.469 inf inf ATOM 153 O H2O 153 149.734−24.594 71.448 inf inf ATOM 154 O H2O 154 149.268 −24.225 71.124 inf infATOM 155 O H2O 155 142.731 −23.841 71.448 inf inf ATOM 156 O H2O 156142.812 −23.520 71.368 inf inf ATOM 157 O H2O 157 149.748 −23.478 71.434inf inf ATOM 158 O H2O 158 147.423 −23.050 71.114 inf inf ATOM 159 O H2O159 148.867 −23.117 71.149 inf inf ATOM 160 O H2O 160 143.329 −22.76471.216 inf inf ATOM 161 O H2O 161 147.365 −22.759 71.245 inf inf ATOM162 O H2O 162 148.847 −22.748 71.181 inf inf ATOM 163 O H2O 163 143.692−22.585 71.396 inf inf ATOM 164 O H2O 164 147.183 −22.382 71.418 inf infATOM 165 O H2O 165 148.288 −22.374 71.403 inf inf ATOM 166 O H2O 166149.548 −22.416 71.339 inf inf ATOM 167 O H2O 167 144.299 −22.076 71.413inf inf ATOM 168 O H2O 168 146.991 −22.215 71.443 inf inf ATOM 169 O H2O169 149.576 −22.203 71.431 inf inf ATOM 170 O H2O 170 145.001 −21.69471.453 inf inf ATOM 171 O H2O 171 146.443 −21.649 71.443 inf inf ATOM172 O H2O 172 145.894 −21.452 71.403 inf inf ATOM 173 O H2O 173 143.692−27.877 71.623 inf inf ATOM 174 O H2O 174 144.406 −27.883 71.619 inf infATOM 175 O H2O 175 144.818 −27.779 71.750 inf inf ATOM 176 O H2O 176142.717 −27.204 71.769 inf inf ATOM 177 O H2O 177 145.720 −27.202 71.768inf inf ATOM 178 O H2O 178 145.935 −27.086 71.728 inf inf ATOM 179 O H2O179 147.001 −26.972 71.791 inf inf ATOM 180 O H2O 180 148.495 −27.05471.756 inf inf ATOM 181 O H2O 181 142.384 −26.441 71.781 inf inf ATOM182 O H2O 182 142.297 −26.096 71.743 inf inf ATOM 183 O H2O 183 149.650−25.913 71.745 inf inf ATOM 184 O H2O 184 149.804 −25.332 71.759 inf infATOM 185 O H2O 185 142.522 −24.758 71.783 inf inf ATOM 186 O H2O 186150.044 −24.594 71.845 inf inf ATOM 187 O H2O 187 142.920 −23.846 71.616inf inf ATOM 188 O H2O 188 150.094 −23.840 71.811 inf inf ATOM 189 O H2O189 150.140 −23.487 71.781 inf inf ATOM 190 O H2O 190 149.996 −23.12871.524 inf inf ATOM 191 O H2O 191 143.870 −22.754 71.775 inf inf ATOM192 O H2O 192 144.036 −22.552 71.787 inf inf ATOM 193 O H2O 193 148.080−22.338 71.501 inf inf ATOM 194 O H2O 194 147.010 −21.997 71.566 inf infATOM 195 O H2O 195 148.458 −21.971 71.538 inf inf ATOM 196 O H2O 196149.817 −21.962 71.710 inf inf ATOM 197 O H2O 197 144.643 −21.670 71.794inf inf ATOM 198 O H2O 198 147.377 −21.815 71.758 inf inf ATOM 199 O H2O199 148.660 −21.627 71.771 inf inf ATOM 200 O H2O 200 149.604 −21.77871.734 inf inf ATOM 201 O H2O 201 145.510 −21.250 71.547 inf inf ATOM202 O H2O 202 146.868 −21.251 71.710 inf inf ATOM 203 O H2O 203 145.161−21.090 71.791 inf inf ATOM 204 O H2O 204 146.261 −20.905 71.603 inf infATOM 205 O H2O 205 145.710 −20.536 71.791 inf inf ATOM 206 O H2O 206146.621 −20.740 71.815 inf inf ATOM 207 O H2O 207 143.707 −28.248 72.013inf inf ATOM 208 O H2O 208 144.405 −28.256 71.996 inf inf ATOM 209 O H2O209 143.294 −27.935 71.947 inf inf ATOM 210 O H2O 210 142.946 −27.72972.153 inf inf ATOM 211 O H2O 211 145.390 −27.597 72.111 inf inf ATOM212 O H2O 212 145.884 −27.333 72.171 inf inf ATOM 213 O H2O 213 147.742−27.170 71.967 inf inf ATOM 214 O H2O 214 142.440 −26.773 72.151 inf infATOM 215 O H2O 215 147.002 −27.056 72.135 inf inf ATOM 216 O H2O 216149.074 −26.861 72.124 inf inf ATOM 217 O H2O 217 149.521 −26.560 72.216inf inf ATOM 218 O H2O 218 142.208 −26.069 71.967 inf inf ATOM 219 O H2O219 142.199 −25.701 71.966 inf inf ATOM 220 O H2O 220 142.187 −25.51572.147 inf inf ATOM 221 O H2O 221 142.397 −24.970 72.151 inf inf ATOM222 O H2O 222 142.720 −24.572 72.153 inf inf ATOM 223 O H2O 223 143.061−24.180 72.171 inf inf ATOM 224 O H2O 224 143.358 −23.534 71.918 inf infATOM 225 O H2O 225 150.315 −23.667 72.155 inf inf ATOM 226 O H2O 226143.910 −23.165 72.103 inf inf ATOM 227 O H2O 227 144.088 −22.957 72.119inf inf ATOM 228 O H2O 228 144.267 −22.388 72.138 inf inf ATOM 229 O H2O229 144.380 −22.178 72.162 inf inf ATOM 230 O H2O 230 150.348 −22.18272.139 inf inf ATOM 231 O H2O 231 148.108 −21.617 71.951 inf inf ATOM232 O H2O 232 150.013 −21.767 72.104 inf inf ATOM 233 O H2O 233 147.340−21.307 72.000 inf inf ATOM 234 O H2O 234 148.473 −21.440 72.140 inf infATOM 235 O H2O 235 144.704 −20.904 72.218 inf inf ATOM 236 O H2O 236147.177 −20.908 72.158 inf inf ATOM 237 O H2O 237 145.147 −20.533 71.955inf inf ATOM 238 O H2O 238 146.825 −20.525 72.144 inf inf ATOM 239 O H2O239 144.833 −20.164 72.106 inf inf ATOM 240 O H2O 240 146.241 −20.18972.032 inf inf ATOM 241 O H2O 241 144.952 −19.783 72.107 inf inf ATOM242 O H2O 242 146.216 −19.842 72.107 inf inf ATOM 243 O H2O 243 145.525−19.468 72.091 inf inf ATOM 244 O H2O 244 145.524 −19.285 72.215 inf infATOM 245 O H2O 245 144.048 −28.821 72.532 inf inf ATOM 246 O H2O 246144.620 −28.691 72.489 inf inf ATOM 247 O H2O 247 144.840 −28.339 72.255inf inf ATOM 248 O H2O 248 145.273 −28.245 72.573 inf inf ATOM 249 O H2O249 145.206 −27.957 72.285 inf inf ATOM 250 O H2O 250 145.561 −27.77972.473 inf inf ATOM 251 O H2O 251 142.595 −27.218 72.480 inf inf ATOM252 O H2O 252 146.633 −27.181 72.334 inf inf ATOM 253 O H2O 253 147.370−27.155 72.339 inf inf ATOM 254 O H2O 254 142.416 −26.796 72.520 inf infATOM 255 O H2O 255 149.241 −26.847 72.309 inf inf ATOM 256 O H2O 256149.756 −26.795 72.547 inf inf ATOM 257 O H2O 257 150.146 −26.445 72.502inf inf ATOM 258 O H2O 258 150.259 −26.038 72.429 inf inf ATOM 259 O H2O259 150.293 −25.686 72.382 inf inf ATOM 260 O H2O 260 150.311 −25.33272.353 inf inf ATOM 261 O H2O 261 150.496 −24.963 72.533 inf inf ATOM262 O H2O 262 150.406 −24.631 72.557 inf inf ATOM 263 O H2O 263 150.332−24.408 72.517 inf inf ATOM 264 O H2O 264 150.307 −23.852 72.338 inf infATOM 265 O H2O 265 143.671 −23.664 72.523 inf inf ATOM 266 O H2O 266144.054 −23.308 72.517 inf inf ATOM 267 O H2O 267 150.636 −22.748 72.488inf inf ATOM 268 O H2O 268 150.564 −22.365 72.506 inf inf ATOM 269 O H2O269 144.546 −21.640 72.520 inf inf ATOM 270 O H2O 270 144.506 −21.29572.521 inf inf ATOM 271 O H2O 271 148.847 −21.270 72.335 inf inf ATOM272 O H2O 272 149.923 −21.503 72.536 inf inf ATOM 273 O H2O 273 147.750−21.063 72.513 inf inf ATOM 274 O H2O 274 149.215 −21.213 72.561 inf infATOM 275 O H2O 275 144.701 −20.533 72.216 inf inf ATOM 276 O H2O 276144.291 −20.164 72.559 inf inf ATOM 277 O H2O 277 147.001 −20.349 72.520inf inf ATOM 278 O H2O 278 146.596 −19.809 72.371 inf inf ATOM 279 O H2O279 144.782 −19.424 72.329 inf inf ATOM 280 O H2O 280 146.486 −19.39572.481 inf inf ATOM 281 O H2O 281 145.159 −19.062 72.347 inf inf ATOM282 O H2O 282 146.294 −19.210 72.473 inf inf ATOM 283 O H2O 283 145.525−18.765 72.451 inf inf ATOM 284 O H2O 284 145.524 −18.548 72.587 inf infATOM 285 O H2O 285 143.655 −28.924 72.853 inf inf ATOM 286 O H2O 286144.789 −28.850 72.884 inf inf ATOM 287 O H2O 287 142.895 −28.315 72.675inf inf ATOM 288 O H2O 288 145.572 −27.954 72.657 inf inf ATOM 289 O H2O289 142.485 −27.547 72.922 inf inf ATOM 290 O H2O 290 146.244 −27.68372.938 inf inf ATOM 291 O H2O 291 146.672 −27.456 72.863 inf inf ATOM292 O H2O 292 147.551 −27.151 72.889 inf inf ATOM 293 O H2O 293 148.476−27.172 72.705 inf inf ATOM 294 O H2O 294 149.218 −27.105 72.738 inf infATOM 295 O H2O 295 148.109 −27.148 72.891 inf inf ATOM 296 O H2O 296149.954 −27.067 72.816 inf inf ATOM 297 O H2O 297 142.307 −26.459 72.889inf inf ATOM 298 O H2O 298 150.882 −26.444 72.884 inf inf ATOM 299 O H2O299 151.038 −26.047 72.773 inf inf ATOM 300 O H2O 300 142.238 −25.70172.926 inf inf ATOM 301 O H2O 301 142.319 −25.313 72.918 inf inf ATOM302 O H2O 302 142.449 −25.005 72.868 inf inf ATOM 303 O H2O 303 142.596−24.804 72.879 inf inf ATOM 304 O H2O 304 142.983 −24.473 72.868 inf infATOM 305 O H2O 305 150.375 −24.223 72.681 inf inf ATOM 306 O H2O 306143.829 −23.829 72.923 inf inf ATOM 307 O H2O 307 144.187 −23.457 72.919inf inf ATOM 308 O H2O 308 144.433 −22.753 72.702 inf inf ATOM 309 O H2O309 144.507 −22.378 72.891 inf inf ATOM 310 O H2O 310 144.506 −21.64072.865 inf inf ATOM 311 O H2O 311 149.407 −21.230 72.889 inf inf ATOM312 O H2O 312 144.269 −20.878 72.901 inf inf ATOM 313 O H2O 313 148.093−21.141 72.906 inf inf ATOM 314 O H2O 314 144.164 −20.553 72.860 inf infATOM 315 O H2O 315 147.019 −20.158 72.693 inf inf ATOM 316 O H2O 316147.029 −19.967 72.873 inf inf ATOM 317 O H2O 317 146.823 −19.421 72.885inf inf ATOM 318 O H2O 318 146.568 −19.105 72.785 inf inf ATOM 319 O H2O319 144.769 −18.654 72.654 inf inf ATOM 320 O H2O 320 146.585 −18.90072.927 inf inf ATOM 321 O H2O 321 144.967 −18.297 72.871 inf inf ATOM322 O H2O 322 146.251 −18.515 72.901 inf inf ATOM 323 O H2O 323 143.679−29.093 73.301 inf inf ATOM 324 O H2O 324 144.048 −29.055 73.069 inf infATOM 325 O H2O 325 144.770 −28.976 73.106 inf inf ATOM 326 O H2O 326142.958 −28.633 73.087 inf inf ATOM 327 O H2O 327 145.380 −28.694 73.227inf inf ATOM 328 O H2O 328 145.577 −28.329 73.048 inf inf ATOM 329 O H2O329 142.521 −27.931 73.052 inf inf ATOM 330 O H2O 330 142.378 −27.54773.255 inf inf ATOM 331 O H2O 331 146.704 −27.506 73.258 inf inf ATOM332 O H2O 332 148.291 −27.190 73.258 inf inf ATOM 333 O H2O 333 149.222−27.345 73.264 inf inf ATOM 334 O H2O 334 149.954 −27.252 72.999 inf infATOM 335 O H2O 335 142.337 −26.809 73.259 inf inf ATOM 336 O H2O 336150.742 −26.848 73.025 inf inf ATOM 337 O H2O 337 151.074 −26.450 73.061inf inf ATOM 338 O H2O 338 151.404 −26.061 73.092 inf inf ATOM 339 O H2O339 151.452 −25.701 73.059 inf inf ATOM 340 O H2O 340 151.507 −25.36873.295 inf inf ATOM 341 O H2O 341 151.051 −24.974 73.084 inf inf ATOM342 O H2O 342 142.913 −24.761 73.277 inf inf ATOM 343 O H2O 343 151.019−24.821 73.275 inf inf ATOM 344 O H2O 344 143.838 −24.201 73.278 inf infATOM 345 O H2O 345 144.025 −24.018 73.276 inf inf ATOM 346 O H2O 346150.577 −23.486 73.276 inf inf ATOM 347 O H2O 347 150.615 −23.144 73.285inf inf ATOM 348 O H2O 348 150.557 −22.367 73.271 inf inf ATOM 349 O H2O349 150.114 −21.665 73.249 inf inf ATOM 350 O H2O 350 144.393 −21.27873.063 inf inf ATOM 351 O H2O 351 144.186 −20.933 73.243 inf inf ATOM352 O H2O 352 148.455 −21.162 73.234 inf inf ATOM 353 O H2O 353 143.997−20.489 73.302 inf inf ATOM 354 O H2O 354 147.700 −20.766 73.287 inf infATOM 355 O H2O 355 147.358 −20.355 73.264 inf inf ATOM 356 O H2O 356147.111 −19.822 73.284 inf inf ATOM 357 O H2O 357 147.031 −19.598 73.250inf inf ATOM 358 O H2O 358 144.017 −18.857 73.243 inf inf ATOM 359 O H2O359 144.347 −18.433 73.203 inf inf ATOM 360 O H2O 360 146.418 −18.34273.276 inf inf ATOM 361 O H2O 361 145.524 −18.014 73.198 inf inf ATOM362 O H2O 362 143.104 −29.065 73.593 inf inf ATOM 363 O H2O 363 144.417−29.211 73.626 inf inf ATOM 364 O H2O 364 145.333 −29.010 73.634 inf infATOM 365 O H2O 365 142.896 −28.894 73.572 inf inf ATOM 366 O H2O 366142.329 −28.332 73.570 inf inf ATOM 367 O H2O 367 142.209 −28.100 73.633inf inf ATOM 368 O H2O 368 142.135 −27.587 73.705 inf inf ATOM 369 O H2O369 146.556 −27.657 73.627 inf inf ATOM 370 O H2O 370 149.585 −27.52673.452 inf inf ATOM 371 O H2O 371 142.225 −27.366 73.640 inf inf ATOM372 O H2O 372 147.329 −27.239 73.631 inf inf ATOM 373 O H2O 373 148.110−27.171 73.444 inf inf ATOM 374 O H2O 374 150.381 −27.477 73.599 inf infATOM 375 O H2O 375 142.298 −26.809 73.606 inf inf ATOM 376 O H2O 376151.190 −26.771 73.646 inf inf ATOM 377 O H2O 377 151.474 −26.274 73.627inf inf ATOM 378 O H2O 378 142.560 −25.327 73.448 inf inf ATOM 379 O H2O379 142.899 −24.929 73.464 inf inf ATOM 380 O H2O 380 151.445 −25.13873.627 inf inf ATOM 381 O H2O 381 143.651 −24.539 73.491 inf inf ATOM382 O H2O 382 144.023 −24.206 73.468 inf inf ATOM 383 O H2O 383 150.752−24.370 73.668 inf inf ATOM 384 O H2O 384 144.417 −23.671 73.628 inf infATOM 385 O H2O 385 150.501 −23.117 73.625 inf inf ATOM 386 O H2O 386150.448 −22.399 73.607 inf inf ATOM 387 O H2O 387 150.328 −22.007 73.445inf inf ATOM 388 O H2O 388 149.971 −21.620 73.455 inf inf ATOM 389 O H2O389 148.882 −21.317 73.662 inf inf ATOM 390 O H2O 390 149.581 −21.46773.620 inf inf ATOM 391 O H2O 391 148.436 −21.223 73.625 inf inf ATOM392 O H2O 392 147.726 −20.728 73.627 inf inf ATOM 393 O H2O 393 143.766−19.820 73.603 inf inf ATOM 394 O H2O 394 147.031 −19.417 73.430 inf infATOM 395 O H2O 395 147.037 −19.236 73.617 inf inf ATOM 396 O H2O 396144.068 −18.517 73.634 inf inf ATOM 397 O H2O 397 146.682 −18.461 73.619inf inf ATOM 398 O H2O 398 144.965 −17.912 73.617 inf inf ATOM 399 O H2O399 146.060 −17.991 73.640 inf inf ATOM 400 O H2O 400 146.632 −33.22774.059 inf inf ATOM 401 O H2O 401 145.905 −32.888 74.020 inf inf ATOM402 O H2O 402 146.279 −32.707 73.869 inf inf ATOM 403 O H2O 403 147.184−32.714 73.999 inf inf ATOM 404 O H2O 404 146.632 −32.346 73.829 inf infATOM 405 O H2O 405 146.053 −31.960 73.939 inf inf ATOM 406 O H2O 406147.180 −31.981 74.010 inf inf ATOM 407 O H2O 407 143.310 −29.504 74.022inf inf ATOM 408 O H2O 408 143.679 −29.392 73.812 inf inf ATOM 409 O H2O409 144.417 −29.333 73.852 inf inf ATOM 410 O H2O 410 145.100 −29.33773.867 inf inf ATOM 411 O H2O 411 142.614 −28.981 73.855 inf inf ATOM412 O H2O 412 145.487 −29.185 74.026 inf inf ATOM 413 O H2O 413 142.255−28.613 73.844 inf inf ATOM 414 O H2O 414 145.933 −28.503 73.976 inf infATOM 415 O H2O 415 146.257 −28.094 74.002 inf inf ATOM 416 O H2O 416146.799 −27.521 74.005 inf inf ATOM 417 O H2O 417 149.597 −27.646 73.997inf inf ATOM 418 O H2O 418 150.489 −27.502 73.997 inf inf ATOM 419 O H2O419 147.350 −27.300 74.017 inf inf ATOM 420 O H2O 420 147.920 −27.14973.996 inf inf ATOM 421 O H2O 421 150.704 −27.380 73.997 inf inf ATOM422 O H2O 422 148.107 −27.138 73.998 inf inf ATOM 423 O H2O 423 151.377−26.473 73.997 inf inf ATOM 424 O H2O 424 142.586 −25.709 73.804 inf infATOM 425 O H2O 425 142.889 −25.291 73.864 inf inf ATOM 426 O H2O 426143.287 −24.929 73.835 inf inf ATOM 427 O H2O 427 151.393 −25.172 73.984inf inf ATOM 428 O H2O 428 151.028 −24.829 73.979 inf inf ATOM 429 O H2O429 150.643 −24.442 73.980 inf inf ATOM 430 O H2O 430 150.377 −23.89873.952 inf inf ATOM 431 O H2O 431 150.360 −23.487 73.828 inf inf ATOM432 O H2O 432 144.583 −22.748 73.997 inf inf ATOM 433 O H2O 433 150.148−22.375 74.006 inf inf ATOM 434 O H2O 434 150.009 −22.167 74.042 inf infATOM 435 O H2O 435 149.638 −21.773 74.039 inf inf ATOM 436 O H2O 436148.639 −21.306 73.985 inf inf ATOM 437 O H2O 437 147.933 −20.892 73.993inf inf ATOM 438 O H2O 438 144.104 −20.681 73.977 inf inf ATOM 439 O H2O439 147.510 −20.183 73.997 inf inf ATOM 440 O H2O 440 143.701 −19.42673.997 inf inf ATOM 441 O H2O 441 147.048 −19.102 73.997 inf inf ATOM442 O H2O 442 144.040 −18.498 73.997 inf inf ATOM 443 O H2O 443 144.417−18.134 73.997 inf inf ATOM 444 O H2O 444 146.313 −18.058 73.973 inf infATOM 445 O H2O 445 145.525 −17.795 74.005 inf inf ATOM 446 O H2O 446145.950 −33.378 74.276 inf inf ATOM 447 O H2O 447 146.632 −33.439 74.203inf inf ATOM 448 O H2O 448 145.564 −33.239 74.415 inf inf ATOM 449 O H2O449 146.228 −33.152 74.044 inf inf ATOM 450 O H2O 450 147.550 −33.08074.370 inf inf ATOM 451 O H2O 451 147.629 −32.740 74.292 inf inf ATOM452 O H2O 452 147.657 −32.346 74.263 inf inf ATOM 453 O H2O 453 145.534−31.982 74.195 inf inf ATOM 454 O H2O 454 147.742 −32.161 74.363 inf infATOM 455 O H2O 455 146.233 −31.549 74.064 inf inf ATOM 456 O H2O 456147.537 −31.618 74.380 inf inf ATOM 457 O H2O 457 146.263 −31.252 74.213inf inf ATOM 458 O H2O 458 147.360 −31.433 74.376 inf inf ATOM 459 O H2O459 146.079 −30.868 74.361 inf inf ATOM 460 O H2O 460 145.894 −30.68874.389 inf inf ATOM 461 O H2O 461 144.803 −29.908 74.419 inf inf ATOM462 O H2O 462 145.517 −29.942 74.392 inf inf ATOM 463 O H2O 463 142.742−29.414 74.359 inf inf ATOM 464 O H2O 464 144.038 −29.641 74.329 inf infATOM 465 O H2O 465 142.522 −29.258 74.346 inf inf ATOM 466 O H2O 466142.029 −28.647 74.369 inf inf ATOM 467 O H2O 467 141.888 −28.318 74.333inf inf ATOM 468 O H2O 468 141.828 −28.102 74.364 inf inf ATOM 469 O H2O469 141.799 −27.597 74.416 inf inf ATOM 470 O H2O 470 146.939 −27.65574.412 inf inf ATOM 471 O H2O 471 149.393 −27.598 74.366 inf inf ATOM472 O H2O 472 141.868 −27.378 74.376 inf inf ATOM 473 O H2O 473 148.109−27.149 74.185 inf inf ATOM 474 O H2O 474 149.148 −27.501 74.404 inf infATOM 475 O H2O 475 142.122 −26.878 74.401 inf inf ATOM 476 O H2O 476142.357 −26.423 74.362 inf inf ATOM 477 O H2O 477 151.408 −26.071 74.406inf inf ATOM 478 O H2O 478 151.467 −25.701 74.197 inf inf ATOM 479 O H2O479 151.398 −25.349 74.165 inf inf ATOM 480 O H2O 480 151.142 −25.09174.387 inf inf ATOM 481 O H2O 481 143.987 −24.717 74.415 inf inf ATOM482 O H2O 482 150.699 −24.589 74.182 inf inf ATOM 483 O H2O 483 150.364−24.199 74.209 inf inf ATOM 484 O H2O 484 150.263 −23.873 74.140 inf infATOM 485 O H2O 485 150.049 −23.117 74.307 inf inf ATOM 486 O H2O 486149.850 −22.354 74.410 inf inf ATOM 487 O H2O 487 149.741 −22.038 74.354inf inf ATOM 488 O H2O 488 144.429 −21.268 74.180 inf inf ATOM 489 O H2O489 148.486 −21.257 74.184 inf inf ATOM 490 O H2O 490 144.383 −21.10274.366 inf inf ATOM 491 O H2O 491 144.122 −20.597 74.382 inf inf ATOM492 O H2O 492 143.992 −20.386 74.384 inf inf ATOM 493 O H2O 493 147.400−19.967 74.366 inf inf ATOM 494 O H2O 494 147.102 −19.084 74.392 inf infATOM 495 O H2O 495 144.063 −18.513 74.361 inf inf ATOM 496 O H2O 496144.606 −17.960 74.369 inf inf ATOM 497 O H2O 497 144.816 −17.851 74.396inf inf ATOM 498 O H2O 498 146.240 −17.802 74.395 inf inf ATOM 499 O H2O499 146.631 −33.922 74.791 inf inf ATOM 500 O H2O 500 147.185 −33.82174.737 inf inf ATOM 501 O H2O 501 145.852 −33.700 74.693 inf inf ATOM502 O H2O 502 145.259 −33.138 74.681 inf inf ATOM 503 O H2O 503 145.124−32.910 74.714 inf inf ATOM 504 O H2O 504 147.981 −32.716 74.697 inf infATOM 505 O H2O 505 144.951 −31.984 74.721 inf inf ATOM 506 O H2O 506144.918 −31.629 74.662 inf inf ATOM 507 O H2O 507 144.780 −31.424 74.728inf inf ATOM 508 O H2O 508 147.361 −31.248 74.560 inf inf ATOM 509 O H2O509 144.868 −30.868 74.673 inf inf ATOM 510 O H2O 510 146.643 −30.85374.512 inf inf ATOM 511 O H2O 511 144.618 −30.492 74.760 inf inf ATOM512 O H2O 512 145.919 −30.476 74.452 inf inf ATOM 513 O H2O 513 146.961−30.715 74.785 inf inf ATOM 514 O H2O 514 144.787 −30.130 74.553 inf infATOM 515 O H2O 515 146.222 −30.173 74.672 inf inf ATOM 516 O H2O 516143.514 −29.717 74.735 inf inf ATOM 517 O H2O 517 145.875 −29.762 74.579inf inf ATOM 518 O H2O 518 142.953 −29.549 74.735 inf inf ATOM 519 O H2O519 142.311 −29.099 74.735 inf inf ATOM 520 O H2O 520 142.149 −28.87674.735 inf inf ATOM 521 O H2O 521 141.846 −28.328 74.735 inf inf ATOM522 O H2O 522 146.679 −27.955 74.528 inf inf ATOM 523 O H2O 523 147.020−27.761 74.716 inf inf ATOM 524 O H2O 524 149.954 −27.692 74.735 inf infATOM 525 O H2O 525 141.934 −27.136 74.735 inf inf ATOM 526 O H2O 526148.475 −27.324 74.735 inf inf ATOM 527 O H2O 527 150.829 −27.130 74.716inf inf ATOM 528 O H2O 528 142.224 −26.634 74.735 inf inf ATOM 529 O H2O529 151.295 −26.071 74.756 inf inf ATOM 530 O H2O 530 143.022 −25.59874.735 inf inf ATOM 531 O H2O 531 151.176 −25.356 74.711 inf inf ATOM532 O H2O 532 150.884 −24.956 74.740 inf inf ATOM 533 O H2O 533 150.481−24.647 74.708 inf inf ATOM 534 O H2O 534 144.506 −24.172 74.736 inf infATOM 535 O H2O 535 149.803 −23.846 74.749 inf inf ATOM 536 O H2O 536149.764 −23.486 74.733 inf inf ATOM 537 O H2O 537 149.926 −23.117 74.538inf inf ATOM 538 O H2O 538 144.566 −22.379 74.735 inf inf ATOM 539 O H2O539 144.466 −21.675 74.701 inf inf ATOM 540 O H2O 540 144.375 −21.26474.735 inf inf ATOM 541 O H2O 541 148.644 −21.314 74.746 inf inf ATOM542 O H2O 542 148.162 −21.002 74.708 inf inf ATOM 543 O H2O 543 147.801−20.671 74.714 inf inf ATOM 544 O H2O 544 143.884 −19.795 74.726 inf infATOM 545 O H2O 545 147.264 −19.407 74.735 inf inf ATOM 546 O H2O 546147.020 −18.682 74.542 inf inf ATOM 547 O H2O 547 146.925 −18.257 74.689inf inf ATOM 548 O H2O 548 146.626 −17.957 74.555 inf inf ATOM 549 O H2O549 144.985 −17.608 74.749 inf inf ATOM 550 O H2O 550 146.094 −17.54474.714 inf inf ATOM 551 O H2O 551 145.870 −17.469 74.759 inf inf ATOM552 O H2O 552 147.370 −34.334 75.146 inf inf ATOM 553 O H2O 553 145.716−33.812 75.107 inf inf ATOM 554 O H2O 554 147.399 −33.881 74.831 inf infATOM 555 O H2O 555 148.040 −33.789 75.022 inf inf ATOM 556 O H2O 556147.806 −33.504 74.803 inf inf ATOM 557 O H2O 557 145.017 −33.068 75.119inf inf ATOM 558 O H2O 558 144.915 −32.729 75.090 inf inf ATOM 559 O H2O559 148.122 −32.346 74.911 inf inf ATOM 560 O H2O 560 144.785 −31.97774.919 inf inf ATOM 561 O H2O 561 144.709 −31.633 74.842 inf inf ATOM562 O H2O 562 147.630 −31.163 75.079 inf inf ATOM 563 O H2O 563 144.478−30.848 74.960 inf inf ATOM 564 O H2O 564 144.415 −30.501 74.916 inf infATOM 565 O H2O 565 144.093 −30.287 75.119 inf inf ATOM 566 O H2O 566146.818 −30.130 75.103 inf inf ATOM 567 O H2O 567 146.272 −29.759 74.910inf inf ATOM 568 O H2O 568 142.960 −29.518 75.103 inf inf ATOM 569 O H2O569 142.310 −29.100 75.105 inf inf ATOM 570 O H2O 570 142.004 −28.66075.104 inf inf ATOM 571 O H2O 571 146.664 −28.310 74.901 inf inf ATOM572 O H2O 572 141.822 −28.102 75.104 inf inf ATOM 573 O H2O 573 141.816−27.549 75.102 inf inf ATOM 574 O H2O 574 149.400 −27.548 75.104 inf infATOM 575 O H2O 575 150.158 −27.593 75.117 inf inf ATOM 576 O H2O 576148.118 −27.407 75.105 inf inf ATOM 577 O H2O 577 150.390 −27.463 75.138inf inf ATOM 578 O H2O 578 150.738 −27.013 75.131 inf inf ATOM 579 O H2O579 142.365 −26.431 75.110 inf inf ATOM 580 O H2O 580 142.493 −26.19975.126 inf inf ATOM 581 O H2O 581 142.936 −25.693 74.921 inf inf ATOM582 O H2O 582 151.072 −25.884 75.113 inf inf ATOM 583 O H2O 583 143.613−25.401 75.153 inf inf ATOM 584 O H2O 584 144.049 −24.964 74.919 inf infATOM 585 O H2O 585 150.713 −25.122 75.129 inf inf ATOM 586 O H2O 586150.310 −24.801 75.086 inf inf ATOM 587 O H2O 587 149.795 −24.215 75.122inf inf ATOM 588 O H2O 588 149.704 −23.873 75.086 inf inf ATOM 589 O H2O589 149.724 −23.104 75.098 inf inf ATOM 590 O H2O 590 149.695 −22.39075.104 inf inf ATOM 591 O H2O 591 144.395 −21.639 75.104 inf inf ATOM592 O H2O 592 144.381 −21.272 75.104 inf inf ATOM 593 O H2O 593 144.325−20.875 75.104 inf inf ATOM 594 O H2O 594 148.815 −21.186 75.170 inf infATOM 595 O H2O 595 148.056 −20.769 75.125 inf inf ATOM 596 O H2O 596147.678 −20.390 75.125 inf inf ATOM 597 O H2O 597 147.404 −19.965 75.104inf inf ATOM 598 O H2O 598 144.005 −19.056 75.085 inf inf ATOM 599 O H2O599 144.069 −18.647 75.104 inf inf ATOM 600 O H2O 600 147.102 −18.34775.105 inf inf ATOM 601 O H2O 601 146.996 −18.137 75.104 inf inf ATOM602 O H2O 602 146.281 −17.548 74.905 inf inf ATOM 603 O H2O 603 145.524−17.371 75.098 inf inf ATOM 604 O H2O 604 146.818 −34.556 75.477 inf infATOM 605 O H2O 605 147.558 −34.572 75.466 inf inf ATOM 606 O H2O 606146.609 −34.445 75.427 inf inf ATOM 607 O H2O 607 148.090 −34.346 75.497inf inf ATOM 608 O H2O 608 145.900 −33.998 75.476 inf inf ATOM 609 O H2O609 145.269 −33.524 75.473 inf inf ATOM 610 O H2O 610 144.975 −33.08275.473 inf inf ATOM 611 O H2O 611 144.848 −32.746 75.442 inf inf ATOM612 O H2O 612 144.643 −32.317 75.487 inf inf ATOM 613 O H2O 613 144.495−32.038 75.427 inf inf ATOM 614 O H2O 614 144.393 −31.614 75.278 inf infATOM 615 O H2O 615 144.358 −31.238 75.263 inf inf ATOM 616 O H2O 616147.519 −30.893 75.485 inf inf ATOM 617 O H2O 617 147.394 −30.674 75.466inf inf ATOM 618 O H2O 618 147.112 −30.179 75.497 inf inf ATOM 619 O H2O619 146.848 −29.751 75.452 inf inf ATOM 620 O H2O 620 143.269 −29.65475.474 inf inf ATOM 621 O H2O 621 142.126 −28.951 75.473 inf inf ATOM622 O H2O 622 146.785 −29.022 75.504 inf inf ATOM 623 O H2O 623 141.848−28.328 75.473 inf inf ATOM 624 O H2O 624 141.817 −27.917 75.474 inf infATOM 625 O H2O 625 141.838 −27.732 75.473 inf inf ATOM 626 O H2O 626147.681 −27.640 75.504 inf inf ATOM 627 O H2O 627 142.037 −27.191 75.473inf inf ATOM 628 O H2O 628 149.223 −27.409 75.500 inf inf ATOM 629 O H2O629 150.467 −27.153 75.440 inf inf ATOM 630 O H2O 630 150.709 −26.81475.300 inf inf ATOM 631 O H2O 631 150.755 −26.460 75.350 inf inf ATOM632 O H2O 632 150.774 −26.070 75.404 inf inf ATOM 633 O H2O 633 150.529−25.687 75.523 inf inf ATOM 634 O H2O 634 143.895 −25.381 75.457 inf infATOM 635 O H2O 635 144.281 −24.995 75.457 inf inf ATOM 636 O H2O 636150.304 −25.186 75.416 inf inf ATOM 637 O H2O 637 144.572 −24.581 75.482inf inf ATOM 638 O H2O 638 149.655 −24.267 75.432 inf inf ATOM 639 O H2O639 149.624 −23.854 75.306 inf inf ATOM 640 O H2O 640 144.748 −23.12175.471 inf inf ATOM 641 O H2O 641 144.627 −22.368 75.472 inf inf ATOM642 O H2O 642 144.408 −21.643 75.290 inf inf ATOM 643 O H2O 643 144.410−21.271 75.448 inf inf ATOM 644 O H2O 644 144.365 −20.899 75.428 inf infATOM 645 O H2O 645 144.304 −20.510 75.449 inf inf ATOM 646 O H2O 646147.562 −20.160 75.471 inf inf ATOM 647 O H2O 647 147.421 −19.839 75.516inf inf ATOM 648 O H2O 648 147.357 −19.612 75.477 inf inf ATOM 649 O H2O649 144.035 −18.870 75.473 inf inf ATOM 650 O H2O 650 147.106 −18.34575.473 inf inf ATOM 651 O H2O 651 144.492 −17.840 75.473 inf inf ATOM652 O H2O 652 146.662 −17.735 75.473 inf inf ATOM 653 O H2O 653 146.234−17.479 75.473 inf inf ATOM 654 O H2O 654 146.992 −34.777 75.829 inf infATOM 655 O H2O 655 147.949 −34.619 75.826 inf inf ATOM 656 O H2O 656148.430 −34.154 75.686 inf inf ATOM 657 O H2O 657 145.844 −34.055 75.825inf inf ATOM 658 O H2O 658 148.787 −33.485 75.779 inf inf ATOM 659 O H2O659 144.868 −32.773 75.857 inf inf ATOM 660 O H2O 660 148.497 −32.33175.646 inf inf ATOM 661 O H2O 661 144.392 −31.991 75.654 inf inf ATOM662 O H2O 662 147.996 −31.560 75.843 inf inf ATOM 663 O H2O 663 144.148−30.891 75.842 inf inf ATOM 664 O H2O 664 143.945 −30.444 75.842 inf infATOM 665 O H2O 665 143.776 −30.184 75.817 inf inf ATOM 666 O H2O 666143.630 −29.990 75.826 inf inf ATOM 667 O H2O 667 142.958 −29.536 75.842inf inf ATOM 668 O H2O 668 142.129 −28.950 75.843 inf inf ATOM 669 O H2O669 141.970 −28.675 75.856 inf inf ATOM 670 O H2O 670 147.153 −28.26375.853 inf inf ATOM 671 O H2O 671 147.325 −28.056 75.861 inf inf ATOM672 O H2O 672 147.712 −27.691 75.856 inf inf ATOM 673 O H2O 673 148.476−27.460 75.818 inf inf ATOM 674 O H2O 674 149.961 −27.191 75.678 inf infATOM 675 O H2O 675 142.372 −26.794 75.848 inf inf ATOM 676 O H2O 676142.584 −26.450 75.650 inf inf ATOM 677 O H2O 677 150.311 −26.440 75.639inf inf ATOM 678 O H2O 678 150.090 −26.071 75.769 inf inf ATOM 679 O H2O679 143.513 −25.730 75.833 inf inf ATOM 680 O H2O 680 150.312 −25.70775.618 inf inf ATOM 681 O H2O 681 144.146 −25.246 75.842 inf inf ATOM682 O H2O 682 144.326 −25.024 75.842 inf inf ATOM 683 O H2O 683 144.491−24.830 75.841 inf inf ATOM 684 O H2O 684 144.722 −24.243 75.841 inf infATOM 685 O H2O 685 149.443 −23.868 75.861 inf inf ATOM 686 O H2O 686144.697 −23.119 75.869 inf inf ATOM 687 O H2O 687 149.811 −22.759 75.842inf inf ATOM 688 O H2O 688 149.741 −22.018 75.846 inf inf ATOM 689 O H2O689 149.513 −21.561 75.842 inf inf ATOM 690 O H2O 690 149.330 −21.34275.842 inf inf ATOM 691 O H2O 691 148.497 −20.868 75.653 inf inf ATOM692 O H2O 692 144.367 −20.488 75.838 inf inf ATOM 693 O H2O 693 148.425−20.796 75.842 inf inf ATOM 694 O H2O 694 144.320 −19.765 75.813 inf infATOM 695 O H2O 695 147.417 −19.590 75.829 inf inf ATOM 696 O H2O 696144.156 −18.666 75.875 inf inf ATOM 697 O H2O 697 144.426 −17.957 75.656inf inf ATOM 698 O H2O 698 146.936 −18.177 75.821 inf inf ATOM 699 O H2O699 146.605 −17.792 75.832 inf inf ATOM 700 O H2O 700 146.213 −17.54375.793 inf inf ATOM 701 O H2O 701 146.623 −34.767 76.211 inf inf ATOM702 O H2O 702 148.122 −34.581 76.020 inf inf ATOM 703 O H2O 703 145.815−34.121 76.211 inf inf ATOM 704 O H2O 704 145.659 −33.855 76.211 inf infATOM 705 O H2O 705 148.870 −33.460 76.023 inf inf ATOM 706 O H2O 706145.207 −33.235 76.194 inf inf ATOM 707 O H2O 707 148.825 −32.721 76.030inf inf ATOM 708 O H2O 708 148.691 −32.327 76.211 inf inf ATOM 709 O H2O709 148.344 −31.915 76.201 inf inf ATOM 710 O H2O 710 148.157 −31.74476.212 inf inf ATOM 711 O H2O 711 147.595 −30.852 76.200 inf inf ATOM712 O H2O 712 147.486 −30.524 76.233 inf inf ATOM 713 O H2O 713 147.243−30.093 76.189 inf inf ATOM 714 O H2O 714 143.305 −29.767 76.026 inf infATOM 715 O H2O 715 146.980 −29.393 76.031 inf inf ATOM 716 O H2O 716142.388 −29.022 76.210 inf inf ATOM 717 O H2O 717 142.182 −28.852 76.218inf inf ATOM 718 O H2O 718 147.259 −28.334 76.162 inf inf ATOM 719 O H2O719 147.597 −27.958 76.177 inf inf ATOM 720 O H2O 720 148.097 −27.69676.236 inf inf ATOM 721 O H2O 721 148.836 −27.339 76.212 inf inf ATOM722 O H2O 722 142.252 −27.043 76.192 inf inf ATOM 723 O H2O 723 149.900−26.768 75.960 inf inf ATOM 724 O H2O 724 143.063 −26.364 76.249 inf infATOM 725 O H2O 725 143.278 −26.032 76.046 inf inf ATOM 726 O H2O 726149.874 −26.071 75.948 inf inf ATOM 727 O H2O 727 149.486 −25.674 76.261inf inf ATOM 728 O H2O 728 144.192 −25.304 76.225 inf inf ATOM 729 O H2O729 149.320 −24.989 76.152 inf inf ATOM 730 O H2O 730 144.672 −24.23576.231 inf inf ATOM 731 O H2O 731 144.647 −23.480 76.225 inf inf ATOM732 O H2O 732 149.710 −23.100 76.203 inf inf ATOM 733 O H2O 733 149.803−22.379 76.220 inf inf ATOM 734 O H2O 734 149.725 −22.023 76.205 inf infATOM 735 O H2O 735 144.340 −21.302 76.241 inf inf ATOM 736 O H2O 736148.651 −20.928 76.211 inf inf ATOM 737 O H2O 737 148.114 −20.525 76.023inf inf ATOM 738 O H2O 738 144.448 −20.352 76.211 inf inf ATOM 739 O H2O739 148.028 −20.401 76.212 inf inf ATOM 740 O H2O 740 147.745 −19.97776.210 inf inf ATOM 741 O H2O 741 147.478 −19.503 76.211 inf inf ATOM742 O H2O 742 144.293 −18.708 76.171 inf inf ATOM 743 O H2O 743 144.374−18.290 76.056 inf inf ATOM 744 O H2O 744 146.945 −18.347 75.999 inf infATOM 745 O H2O 745 146.641 −17.938 76.034 inf inf ATOM 746 O H2O 746145.525 −17.677 76.274 inf inf ATOM 747 O H2O 747 146.255 −17.777 76.201inf inf ATOM 748 O H2O 748 146.623 −34.767 76.581 inf inf ATOM 749 O H2O749 148.054 −34.664 76.581 inf inf ATOM 750 O H2O 750 148.453 −34.35276.580 inf inf ATOM 751 O H2O 751 145.414 −33.406 76.555 inf inf ATOM752 O H2O 752 145.148 −33.088 76.397 inf inf ATOM 753 O H2O 753 145.079−32.953 76.581 inf inf ATOM 754 O H2O 754 148.690 −32.327 76.581 inf infATOM 755 O H2O 755 148.383 −31.887 76.549 inf inf ATOM 756 O H2O 756148.034 −31.532 76.548 inf inf ATOM 757 O H2O 757 144.168 −30.897 76.599inf inf ATOM 758 O H2O 758 144.049 −30.684 76.580 inf inf ATOM 759 O H2O759 143.736 −30.259 76.581 inf inf ATOM 760 O H2O 760 143.396 −29.83376.580 inf inf ATOM 761 O H2O 761 143.023 −29.443 76.611 inf inf ATOM762 O H2O 762 142.727 −29.055 76.593 inf inf ATOM 763 O H2O 763 142.348−28.686 76.620 inf inf ATOM 764 O H2O 764 142.222 −28.465 76.553 inf infATOM 765 O H2O 765 142.089 −27.916 76.533 inf inf ATOM 766 O H2O 766142.120 −27.524 76.455 inf inf ATOM 767 O H2O 767 148.489 −27.567 76.393inf inf ATOM 768 O H2O 768 142.397 −27.188 76.570 inf inf ATOM 769 O H2O769 142.597 −26.841 76.377 inf inf ATOM 770 O H2O 770 149.369 −26.77776.568 inf inf ATOM 771 O H2O 771 149.440 −26.451 76.598 inf inf ATOM772 O H2O 772 149.554 −26.086 76.381 inf inf ATOM 773 O H2O 773 144.090−25.559 76.564 inf inf ATOM 774 O H2O 774 144.383 −25.134 76.590 inf infATOM 775 O H2O 775 149.180 −24.973 76.379 inf inf ATOM 776 O H2O 776144.553 −24.225 76.581 inf inf ATOM 777 O H2O 777 149.187 −24.036 76.572inf inf ATOM 778 O H2O 778 144.436 −23.296 76.593 inf inf ATOM 779 O H2O779 149.632 −23.071 76.534 inf inf ATOM 780 O H2O 780 144.180 −22.41176.518 inf inf ATOM 781 O H2O 781 143.935 −22.010 76.652 inf inf ATOM782 O H2O 782 144.132 −21.640 76.451 inf inf ATOM 783 O H2O 783 144.211−21.266 76.568 inf inf ATOM 784 O H2O 784 144.246 −20.902 76.588 inf infATOM 785 O H2O 785 148.035 −20.455 76.585 inf inf ATOM 786 O H2O 786144.447 −20.164 76.400 inf inf ATOM 787 O H2O 787 144.455 −19.789 76.575inf inf ATOM 788 O H2O 788 144.498 −19.380 76.639 inf inf ATOM 789 O H2O789 147.127 −19.114 76.557 inf inf ATOM 790 O H2O 790 146.773 −18.73176.537 inf inf ATOM 791 O H2O 791 144.956 −18.299 76.615 inf inf ATOM792 O H2O 792 144.842 −18.005 76.340 inf inf ATOM 793 O H2O 793 145.525−17.862 76.483 inf inf ATOM 794 O H2O 794 146.263 −17.949 76.396 inf infATOM 795 O H2O 795 146.992 −34.775 76.962 inf inf ATOM 796 O H2O 796147.948 −34.617 76.966 inf inf ATOM 797 O H2O 797 148.172 −34.471 76.982inf inf ATOM 798 O H2O 798 145.862 −34.028 76.960 inf inf ATOM 799 O H2O799 148.818 −33.634 76.949 inf inf ATOM 800 O H2O 800 144.899 −32.76676.950 inf inf ATOM 801 O H2O 801 144.756 −32.543 76.950 inf inf ATOM802 O H2O 802 148.483 −31.972 76.764 inf inf ATOM 803 O H2O 803 148.146−31.577 76.745 inf inf ATOM 804 O H2O 804 148.013 −31.204 76.896 inf infATOM 805 O H2O 805 143.937 −30.451 76.950 inf inf ATOM 806 O H2O 806143.595 −30.064 76.950 inf inf ATOM 807 O H2O 807 143.414 −29.843 76.949inf inf ATOM 808 O H2O 808 143.066 −29.422 76.951 inf inf ATOM 809 O H2O809 142.766 −29.017 76.946 inf inf ATOM 810 O H2O 810 142.511 −28.65476.813 inf inf ATOM 811 O H2O 811 142.289 −28.246 76.666 inf inf ATOM812 O H2O 812 147.796 −28.495 76.933 inf inf ATOM 813 O H2O 813 148.135−27.948 76.749 inf inf ATOM 814 O H2O 814 142.551 −27.735 76.967 inf infATOM 815 O H2O 815 142.588 −27.190 76.744 inf inf ATOM 816 O H2O 816142.974 −26.850 76.740 inf inf ATOM 817 O H2O 817 149.258 −26.769 76.927inf inf ATOM 818 O H2O 818 149.350 −26.425 76.942 inf inf ATOM 819 O H2O819 143.912 −25.774 76.950 inf inf ATOM 820 O H2O 820 149.159 −25.26876.950 inf inf ATOM 821 O H2O 821 144.495 −24.937 76.949 inf inf ATOM822 O H2O 822 144.511 −24.224 76.927 inf inf ATOM 823 O H2O 823 149.125−23.880 76.977 inf inf ATOM 824 O H2O 824 149.208 −23.668 76.947 inf infATOM 825 O H2O 825 149.409 −23.120 76.953 inf inf ATOM 826 O H2O 826149.465 −22.749 76.993 inf inf ATOM 827 O H2O 827 149.468 −22.379 76.995inf inf ATOM 828 O H2O 828 143.746 −22.010 76.832 inf inf ATOM 829 O H2O829 143.784 −21.615 76.871 inf inf ATOM 830 O H2O 830 149.018 −21.28576.949 inf inf ATOM 831 O H2O 831 148.659 −20.910 76.952 inf inf ATOM832 O H2O 832 148.105 −20.536 76.766 inf inf ATOM 833 O H2O 833 147.919−20.166 76.951 inf inf ATOM 834 O H2O 834 147.639 −19.735 76.949 inf infATOM 835 O H2O 835 147.450 −19.497 76.982 inf inf ATOM 836 O H2O 836144.898 −19.008 76.998 inf inf ATOM 837 O H2O 837 144.832 −18.706 76.719inf inf ATOM 838 O H2O 838 146.438 −18.704 76.938 inf inf ATOM 839 O H2O839 145.525 −18.298 76.793 inf inf ATOM 840 O H2O 840 146.228 −18.56176.903 inf inf ATOM 841 O H2O 841 146.812 −34.588 77.327 inf inf ATOM842 O H2O 842 146.108 −34.161 77.307 inf inf ATOM 843 O H2O 843 148.454−34.172 77.131 inf inf ATOM 844 O H2O 844 148.536 −34.049 77.317 inf infATOM 845 O H2O 845 148.792 −33.615 77.318 inf inf ATOM 846 O H2O 846148.941 −33.083 77.348 inf inf ATOM 847 O H2O 847 144.635 −32.323 77.319inf inf ATOM 848 O H2O 848 144.518 −32.012 77.296 inf inf ATOM 849 O H2O849 144.401 −31.803 77.314 inf inf ATOM 850 O H2O 850 148.131 −31.23277.124 inf inf ATOM 851 O H2O 851 148.136 −30.877 77.372 inf inf ATOM852 O H2O 852 148.155 −30.500 77.365 inf inf ATOM 853 O H2O 853 148.121−30.314 77.312 inf inf ATOM 854 O H2O 854 147.792 −29.736 77.090 inf infATOM 855 O H2O 855 147.791 −29.427 77.362 inf inf ATOM 856 O H2O 856147.740 −29.208 77.319 inf inf ATOM 857 O H2O 857 147.829 −28.619 77.350inf inf ATOM 858 O H2O 858 142.653 −27.916 77.079 inf inf ATOM 859 O H2O859 142.874 −27.514 77.192 inf inf ATOM 860 O H2O 860 148.676 −27.56177.319 inf inf ATOM 861 O H2O 861 149.025 −27.174 77.319 inf inf ATOM862 O H2O 862 143.372 −26.652 77.299 inf inf ATOM 863 O H2O 863 143.592−26.217 77.322 inf inf ATOM 864 O H2O 864 143.956 −25.794 77.319 inf infATOM 865 O H2O 865 149.164 −25.266 77.318 inf inf ATOM 866 O H2O 866149.053 −24.958 77.319 inf inf ATOM 867 O H2O 867 149.028 −24.225 77.319inf inf ATOM 868 O H2O 868 149.073 −23.866 77.320 inf inf ATOM 869 O H2O869 144.044 −23.306 77.317 inf inf ATOM 870 O H2O 870 149.268 −23.11577.271 inf inf ATOM 871 O H2O 871 149.314 −22.747 77.287 inf inf ATOM872 O H2O 872 143.435 −22.010 77.293 inf inf ATOM 873 O H2O 873 149.246−21.694 77.314 inf inf ATOM 874 O H2O 874 149.163 −21.479 77.317 inf infATOM 875 O H2O 875 148.883 −21.062 77.308 inf inf ATOM 876 O H2O 876148.311 −20.516 77.305 inf inf ATOM 877 O H2O 877 147.989 −20.101 77.300inf inf ATOM 878 O H2O 878 147.718 −19.813 77.138 inf inf ATOM 879 O H2O879 147.243 −19.369 77.318 inf inf ATOM 880 O H2O 880 144.989 −19.09477.300 inf inf ATOM 881 O H2O 881 145.521 −18.664 77.155 inf inf ATOM882 O H2O 882 146.278 −18.820 77.341 inf inf ATOM 883 O H2O 883 147.002−34.566 77.680 inf inf ATOM 884 O H2O 884 146.146 −34.124 77.688 inf infATOM 885 O H2O 885 148.103 −34.368 77.688 inf inf ATOM 886 O H2O 886148.519 −34.048 77.688 inf inf ATOM 887 O H2O 887 148.905 −33.411 77.688inf inf ATOM 888 O H2O 888 144.969 −32.717 77.688 inf inf ATOM 889 O H2O889 144.771 −32.543 77.686 inf inf ATOM 890 O H2O 890 144.422 −31.97477.506 inf inf ATOM 891 O H2O 891 148.460 −31.619 77.509 inf inf ATOM892 O H2O 892 148.409 −31.451 77.688 inf inf ATOM 893 O H2O 893 148.290−30.869 77.690 inf inf ATOM 894 O H2O 894 143.726 −30.284 77.688 inf infATOM 895 O H2O 895 143.405 −29.851 77.688 inf inf ATOM 896 O H2O 896143.230 −29.658 77.715 inf inf ATOM 897 O H2O 897 142.944 −29.207 77.687inf inf ATOM 898 O H2O 898 147.962 −28.665 77.672 inf inf ATOM 899 O H2O899 148.180 −28.214 77.729 inf inf ATOM 900 O H2O 900 143.141 −27.55877.683 inf inf ATOM 901 O H2O 901 143.263 −27.343 77.701 inf inf ATOM902 O H2O 902 143.331 −26.822 77.500 inf inf ATOM 903 O H2O 903 143.505−26.444 77.688 inf inf ATOM 904 O H2O 904 143.757 −25.995 77.688 inf infATOM 905 O H2O 905 149.278 −25.703 77.648 inf inf ATOM 906 O H2O 906144.447 −25.160 77.688 inf inf ATOM 907 O H2O 907 149.065 −24.595 77.679inf inf ATOM 908 O H2O 908 144.422 −24.038 77.688 inf inf ATOM 909 O H2O909 143.781 −23.200 77.660 inf inf ATOM 910 O H2O 910 143.469 −22.75977.681 inf inf ATOM 911 O H2O 911 143.357 −22.413 77.654 inf inf ATOM912 O H2O 912 143.290 −22.010 77.742 inf inf ATOM 913 O H2O 913 143.320−21.827 77.691 inf inf ATOM 914 O H2O 914 143.394 −21.246 77.663 inf infATOM 915 O H2O 915 148.996 −20.925 77.714 inf inf ATOM 916 O H2O 916148.691 −20.510 77.659 inf inf ATOM 917 O H2O 917 148.121 −20.150 77.497inf inf ATOM 918 O H2O 918 144.358 −19.894 77.688 inf inf ATOM 919 O H2O919 144.793 −19.431 77.502 inf inf ATOM 920 O H2O 920 147.430 −19.49177.688 inf inf ATOM 921 O H2O 921 145.329 −19.031 77.695 inf inf ATOM922 O H2O 922 145.894 −18.883 77.685 inf inf ATOM 923 O H2O 923 147.370−34.566 77.873 inf inf ATOM 924 O H2O 924 146.969 −34.505 78.089 inf infATOM 925 O H2O 925 148.303 −34.206 78.062 inf inf ATOM 926 O H2O 926148.500 −34.029 78.066 inf inf ATOM 927 O H2O 927 148.797 −33.617 78.043inf inf ATOM 928 O H2O 928 148.960 −33.084 78.047 inf inf ATOM 929 O H2O929 144.545 −32.402 78.057 inf inf ATOM 930 O H2O 930 144.375 −32.19178.057 inf inf ATOM 931 O H2O 931 148.552 −31.689 78.057 inf inf ATOM932 O H2O 932 144.010 −30.818 78.056 inf inf ATOM 933 O H2O 933 148.304−30.500 78.057 inf inf ATOM 934 O H2O 934 143.461 −29.784 78.068 inf infATOM 935 O H2O 935 148.000 −29.374 78.057 inf inf ATOM 936 O H2O 936147.960 −29.024 78.057 inf inf ATOM 937 O H2O 937 142.914 −28.286 78.004inf inf ATOM 938 O H2O 938 142.972 −28.105 78.048 inf inf ATOM 939 O H2O939 143.219 −27.609 78.026 inf inf ATOM 940 O H2O 940 143.423 −27.16278.074 inf inf ATOM 941 O H2O 941 148.874 −27.010 78.079 inf inf ATOM942 O H2O 942 149.092 −26.460 78.097 inf inf ATOM 943 O H2O 943 149.229−26.070 77.878 inf inf ATOM 944 O H2O 944 149.223 −25.701 77.874 inf infATOM 945 O H2O 945 149.226 −25.330 77.871 inf inf ATOM 946 O H2O 946149.183 −24.972 77.888 inf inf ATOM 947 O H2O 947 149.286 −24.589 78.141inf inf ATOM 948 O H2O 948 144.333 −23.791 78.057 inf inf ATOM 949 O H2O949 149.262 −23.856 78.104 inf inf ATOM 950 O H2O 950 143.757 −23.22378.057 inf inf ATOM 951 O H2O 951 149.141 −23.090 78.057 inf inf ATOM952 O H2O 952 143.324 −22.422 78.057 inf inf ATOM 953 O H2O 953 149.200−22.010 77.874 inf inf ATOM 954 O H2O 954 143.308 −21.640 78.058 inf infATOM 955 O H2O 955 143.323 −21.229 78.057 inf inf ATOM 956 O H2O 956149.141 −20.856 77.994 inf inf ATOM 957 O H2O 957 148.869 −20.515 77.859inf inf ATOM 958 O H2O 958 143.903 −20.204 78.057 inf inf ATOM 959 O H2O959 144.102 −20.061 78.057 inf inf ATOM 960 O H2O 960 148.140 −19.90678.036 inf inf ATOM 961 O H2O 961 147.217 −19.379 78.073 inf inf ATOM962 O H2O 962 145.351 −19.082 78.050 inf inf ATOM 963 O H2O 963 145.894−18.942 78.047 inf inf ATOM 964 O H2O 964 146.655 −34.309 78.403 inf infATOM 965 O H2O 965 148.036 −34.269 78.391 inf inf ATOM 966 O H2O 966148.429 −33.959 78.407 inf inf ATOM 967 O H2O 967 148.825 −33.447 78.232inf inf ATOM 968 O H2O 968 148.820 −33.265 78.411 inf inf ATOM 969 O H2O969 148.872 −32.715 78.373 inf inf ATOM 970 O H2O 970 148.828 −32.53378.416 inf inf ATOM 971 O H2O 971 148.613 −32.015 78.397 inf inf ATOM972 O H2O 972 148.486 −31.605 78.247 inf inf ATOM 973 O H2O 973 144.070−31.051 78.426 inf inf ATOM 974 O H2O 974 143.705 −30.118 78.229 inf infATOM 975 O H2O 975 143.713 −29.935 78.403 inf inf ATOM 976 O H2O 976147.955 −29.384 78.440 inf inf ATOM 977 O H2O 977 147.914 −29.024 78.422inf inf ATOM 978 O H2O 978 143.007 −28.305 78.213 inf inf ATOM 979 O H2O979 148.068 −28.084 78.427 inf inf ATOM 980 O H2O 980 143.495 −27.54778.425 inf inf ATOM 981 O H2O 981 148.482 −27.365 78.428 inf inf ATOM982 O H2O 982 148.704 −26.823 78.455 inf inf ATOM 983 O H2O 983 148.825−26.617 78.412 inf inf ATOM 984 O H2O 984 144.087 −25.935 78.397 inf infATOM 985 O H2O 985 144.723 −25.275 78.337 inf inf ATOM 986 O H2O 986149.296 −25.296 78.429 inf inf ATOM 987 O H2O 987 144.803 −24.593 78.216inf inf ATOM 988 O H2O 988 144.966 −24.227 78.431 inf inf ATOM 989 O H2O989 144.615 −23.837 78.404 inf inf ATOM 990 O H2O 990 144.418 −23.67078.426 inf inf ATOM 991 O H2O 991 143.799 −23.182 78.448 inf inf ATOM992 O H2O 992 143.625 −22.969 78.444 inf inf ATOM 993 O H2O 993 143.299−22.010 78.243 inf inf ATOM 994 O H2O 994 143.304 −21.641 78.243 inf infATOM 995 O H2O 995 149.378 −21.275 78.433 inf inf ATOM 996 O H2O 996143.577 −20.589 78.398 inf inf ATOM 997 O H2O 997 143.908 −20.209 78.408inf inf ATOM 998 O H2O 998 144.390 −19.918 78.444 inf inf ATOM 999 O H2O999 148.275 −19.838 78.439 inf inf ATOM 1000 O H2O 1000 147.386 −19.57478.437 inf inf ATOM 1001 O H2O 1001 145.525 −19.060 78.240 inf inf ATOM1002 O H2O 1002 146.298 −19.104 78.495 inf inf ATOM 1003 O H2O 1003146.946 −19.324 78.399 inf inf ATOM 1004 O H2O 1004 147.370 −34.26378.720 inf inf ATOM 1005 O H2O 1005 148.079 −34.133 78.582 inf inf ATOM1006 O H2O 1006 146.972 −34.095 78.883 inf inf ATOM 1007 O H2O 1007148.274 −33.804 78.776 inf inf ATOM 1008 O H2O 1008 148.437 −33.61478.763 inf inf ATOM 1009 O H2O 1009 145.901 −33.261 78.790 inf inf ATOM1010 O H2O 1010 145.204 −32.755 78.747 inf inf ATOM 1011 O H2O 1011144.613 −32.334 78.791 inf inf ATOM 1012 O H2O 1012 144.452 −32.13878.784 inf inf ATOM 1013 O H2O 1013 144.224 −31.610 78.799 inf inf ATOM1014 O H2O 1014 144.082 −30.869 78.588 inf inf ATOM 1015 O H2O 1015144.159 −30.522 78.827 inf inf ATOM 1016 O H2O 1016 148.062 −30.09778.864 inf inf ATOM 1017 O H2O 1017 143.889 −29.753 78.778 inf inf ATOM1018 O H2O 1018 143.845 −29.401 78.815 inf inf ATOM 1019 O H2O 1019143.691 −29.202 78.777 inf inf ATOM 1020 O H2O 1020 147.810 −28.62278.757 inf inf ATOM 1021 O H2O 1021 143.377 −27.943 78.544 inf inf ATOM1022 O H2O 1022 148.024 −28.046 78.767 inf inf ATOM 1023 O H2O 1023148.327 −27.562 78.805 inf inf ATOM 1024 O H2O 1024 148.431 −27.21378.830 inf inf ATOM 1025 O H2O 1025 148.565 −26.776 78.761 inf inf ATOM1026 O H2O 1026 148.805 −26.419 78.590 inf inf ATOM 1027 O H2O 1027148.782 −26.223 78.763 inf inf ATOM 1028 O H2O 1028 144.785 −25.88478.797 inf inf ATOM 1029 O H2O 1029 145.144 −25.326 78.628 inf inf ATOM1030 O H2O 1030 149.355 −25.316 78.810 inf inf ATOM 1031 O H2O 1031145.194 −24.592 78.573 inf inf ATOM 1032 O H2O 1032 145.314 −24.23478.813 inf inf ATOM 1033 O H2O 1033 145.184 −24.023 78.773 inf inf ATOM1034 O H2O 1034 144.804 −23.642 78.773 inf inf ATOM 1035 O H2O 1035144.387 −23.391 78.855 inf inf ATOM 1036 O H2O 1036 143.727 −22.90978.771 inf inf ATOM 1037 O H2O 1037 149.224 −22.381 78.610 inf inf ATOM1038 O H2O 1038 149.312 −21.997 78.795 inf inf ATOM 1039 O H2O 1039149.393 −21.272 78.795 inf inf ATOM 1040 O H2O 1040 143.642 −20.69478.808 inf inf ATOM 1041 O H2O 1041 144.048 −20.165 78.611 inf inf ATOM1042 O H2O 1042 148.936 −20.259 78.795 inf inf ATOM 1043 O H2O 1043144.950 −19.764 78.816 inf inf ATOM 1044 O H2O 1044 145.150 −19.60478.802 inf inf ATOM 1045 O H2O 1045 146.986 −19.456 78.596 inf inf ATOM1046 O H2O 1046 145.549 −19.317 78.745 inf inf ATOM 1047 O H2O 1047146.600 −19.338 78.732 inf inf ATOM 1048 O H2O 1048 147.370 −33.90579.095 inf inf ATOM 1049 O H2O 1049 146.466 −33.434 79.126 inf inf ATOM1050 O H2O 1050 147.747 −33.654 79.189 inf inf ATOM 1051 O H2O 1051145.890 −33.089 78.985 inf inf ATOM 1052 O H2O 1052 148.270 −33.08479.130 inf inf ATOM 1053 O H2O 1053 145.678 −32.761 79.195 inf inf ATOM1054 O H2O 1054 144.772 −32.364 78.991 inf inf ATOM 1055 O H2O 1055148.224 −32.373 79.096 inf inf ATOM 1056 O H2O 1056 144.758 −32.19079.187 inf inf ATOM 1057 O H2O 1057 144.352 −31.567 79.085 inf inf ATOM1058 O H2O 1058 144.326 −31.238 79.102 inf inf ATOM 1059 O H2O 1059147.917 −30.869 79.160 inf inf ATOM 1060 O H2O 1060 144.523 −30.49979.242 inf inf ATOM 1061 O H2O 1061 144.285 −30.110 79.112 inf inf ATOM1062 O H2O 1062 144.244 −29.762 79.153 inf inf ATOM 1063 O H2O 1063144.049 −29.393 78.978 inf inf ATOM 1064 O H2O 1064 143.743 −28.99278.885 inf inf ATOM 1065 O H2O 1065 147.666 −29.023 78.907 inf inf ATOM1066 O H2O 1066 144.421 −28.841 79.149 inf inf ATOM 1067 O H2O 1067143.719 −28.306 78.920 inf inf ATOM 1068 O H2O 1068 147.613 −28.33379.223 inf inf ATOM 1069 O H2O 1069 144.614 −27.916 79.137 inf inf ATOM1070 O H2O 1070 144.082 −27.547 78.929 inf inf ATOM 1071 O H2O 1071148.239 −27.529 79.146 inf inf ATOM 1072 O H2O 1072 148.329 −27.18879.180 inf inf ATOM 1073 O H2O 1073 148.470 −26.807 78.977 inf inf ATOM1074 O H2O 1074 144.784 −26.628 79.192 inf inf ATOM 1075 O H2O 1075144.471 −26.142 78.891 inf inf ATOM 1076 O H2O 1076 145.349 −26.07679.143 inf inf ATOM 1077 O H2O 1077 145.462 −25.652 79.042 inf inf ATOM1078 O H2O 1078 149.042 −25.714 79.167 inf inf ATOM 1079 O H2O 1079149.378 −25.317 79.165 inf inf ATOM 1080 O H2O 1080 145.551 −24.58378.954 inf inf ATOM 1081 O H2O 1081 149.589 −24.593 79.164 inf inf ATOM1082 O H2O 1082 149.592 −24.408 79.165 inf inf ATOM 1083 O H2O 1083149.471 −23.838 79.165 inf inf ATOM 1084 O H2O 1084 149.364 −23.49779.159 inf inf ATOM 1085 O H2O 1085 144.762 −23.337 79.188 inf inf ATOM1086 O H2O 1086 144.067 −22.919 79.141 inf inf ATOM 1087 O H2O 1087143.827 −22.393 79.201 inf inf ATOM 1088 O H2O 1088 143.786 −22.03779.242 inf inf ATOM 1089 O H2O 1089 149.331 −21.640 79.142 inf inf ATOM1090 O H2O 1090 143.684 −21.089 79.161 inf inf ATOM 1091 O H2O 1091149.056 −20.517 79.173 inf inf ATOM 1092 O H2O 1092 144.397 −20.29979.214 inf inf ATOM 1093 O H2O 1093 144.814 −20.072 79.106 inf inf ATOM1094 O H2O 1094 145.474 −19.728 79.097 inf inf ATOM 1095 O H2O 1095147.024 −19.709 79.016 inf inf ATOM 1096 O H2O 1096 147.923 −19.80279.162 inf inf ATOM 1097 O H2O 1097 145.546 −19.480 78.926 inf inf ATOM1098 O H2O 1098 146.263 −19.436 78.970 inf inf ATOM 1099 O H2O 1099146.670 −33.415 79.234 inf inf ATOM 1100 O H2O 1100 146.290 −33.04879.304 inf inf ATOM 1101 O H2O 1101 147.746 −33.091 79.376 inf inf ATOM1102 O H2O 1102 146.287 −32.839 79.474 inf inf ATOM 1103 O H2O 1103147.405 −32.750 79.489 inf inf ATOM 1104 O H2O 1104 145.497 −32.40379.433 inf inf ATOM 1105 O H2O 1105 147.562 −32.349 79.544 inf inf ATOM1106 O H2O 1106 144.977 −31.971 79.521 inf inf ATOM 1107 O H2O 1107147.830 −31.977 79.440 inf inf ATOM 1108 O H2O 1108 147.618 −31.60879.623 inf inf ATOM 1109 O H2O 1109 147.574 −31.229 79.563 inf inf ATOM1110 O H2O 1110 147.546 −30.870 79.520 inf inf ATOM 1111 O H2O 1111144.944 −30.499 79.596 inf inf ATOM 1112 O H2O 1112 144.703 −30.13179.432 inf inf ATOM 1113 O H2O 1113 144.467 −29.762 79.274 inf inf ATOM1114 O H2O 1114 147.355 −29.954 79.512 inf inf ATOM 1115 O H2O 1115144.932 −29.374 79.592 inf inf ATOM 1116 O H2O 1116 147.343 −29.42079.270 inf inf ATOM 1117 O H2O 1117 146.780 −29.024 79.478 inf inf ATOM1118 O H2O 1118 145.106 −28.633 79.423 inf inf ATOM 1119 O H2O 1119146.632 −28.840 79.528 inf inf ATOM 1120 O H2O 1120 144.827 −28.28479.236 inf inf ATOM 1121 O H2O 1121 146.984 −28.453 79.499 inf inf ATOM1122 O H2O 1122 145.147 −27.904 79.390 inf inf ATOM 1123 O H2O 1123147.366 −28.093 79.525 inf inf ATOM 1124 O H2O 1124 145.158 −27.54779.336 inf inf ATOM 1125 O H2O 1125 148.115 −27.551 79.353 inf inf ATOM1126 O H2O 1126 145.449 −27.145 79.458 inf inf ATOM 1127 O H2O 1127144.802 −26.808 79.181 inf inf ATOM 1128 O H2O 1128 148.228 −26.78679.490 inf inf ATOM 1129 O H2O 1129 148.431 −26.417 79.326 inf inf ATOM1130 O H2O 1130 148.451 −26.238 79.525 inf inf ATOM 1131 O H2O 1131146.015 −25.703 79.596 inf inf ATOM 1132 O H2O 1132 145.967 −25.33179.614 inf inf ATOM 1133 O H2O 1133 145.954 −24.963 79.565 inf inf ATOM1134 O H2O 1134 149.578 −24.594 79.348 inf inf ATOM 1135 O H2O 1135149.516 −24.196 79.563 inf inf ATOM 1136 O H2O 1136 145.102 −23.52279.385 inf inf ATOM 1137 O H2O 1137 144.750 −23.173 79.386 inf inf ATOM1138 O H2O 1138 149.221 −23.301 79.535 inf inf ATOM 1139 O H2O 1139144.604 −22.746 79.531 inf inf ATOM 1140 O H2O 1140 144.246 −22.37079.501 inf inf ATOM 1141 O H2O 1141 143.993 −22.038 79.431 inf inf ATOM1142 O H2O 1142 143.732 −21.641 79.296 inf inf ATOM 1143 O H2O 1143149.254 −21.641 79.365 inf inf ATOM 1144 O H2O 1144 149.257 −21.27279.367 inf inf ATOM 1145 O H2O 1145 149.066 −20.891 79.557 inf inf ATOM1146 O H2O 1146 144.408 −20.708 79.553 inf inf ATOM 1147 O H2O 1147145.524 −20.457 79.457 inf inf ATOM 1148 O H2O 1148 144.786 −20.41479.469 inf inf ATOM 1149 O H2O 1149 145.893 −20.163 79.352 inf inf ATOM1150 O H2O 1150 146.630 −20.338 79.547 inf inf ATOM 1151 O H2O 1151148.803 −20.392 79.491 inf inf ATOM 1152 O H2O 1152 147.022 −20.03379.480 inf inf ATOM 1153 O H2O 1153 148.104 −19.992 79.525 inf inf ATOM1154 O H2O 1154 147.001 −32.658 79.633 inf inf ATOM 1155 O H2O 1155147.015 −32.366 79.766 inf inf ATOM 1156 O H2O 1156 145.879 −31.99979.770 inf inf ATOM 1157 O H2O 1157 146.817 −31.979 79.910 inf inf ATOM1158 O H2O 1158 145.145 −31.615 79.743 inf inf ATOM 1159 O H2O 1159146.446 −31.608 79.913 inf inf ATOM 1160 O H2O 1160 144.843 −31.23879.633 inf inf ATOM 1161 O H2O 1161 145.894 −31.422 79.898 inf inf ATOM1162 O H2O 1162 146.632 −31.424 79.899 inf inf ATOM 1163 O H2O 1163145.144 −30.861 79.746 inf inf ATOM 1164 O H2O 1164 146.263 −30.84279.826 inf inf ATOM 1165 O H2O 1165 145.162 −30.500 79.703 inf inf ATOM1166 O H2O 1166 146.632 −30.500 79.769 inf inf ATOM 1167 O H2O 1167145.502 −30.131 79.810 inf inf ATOM 1168 O H2O 1168 146.994 −30.13579.693 inf inf ATOM 1169 O H2O 1169 145.894 −29.762 79.860 inf inf ATOM1170 O H2O 1170 145.159 −29.395 79.710 inf inf ATOM 1171 O H2O 1171146.584 −29.417 79.648 inf inf ATOM 1172 O H2O 1172 146.232 −29.04879.617 inf inf ATOM 1173 O H2O 1173 146.223 −28.646 79.584 inf inf ATOM1174 O H2O 1174 146.633 −28.245 79.609 inf inf ATOM 1175 O H2O 1175146.631 −27.946 79.762 inf inf ATOM 1176 O H2O 1176 146.231 −27.61379.851 inf inf ATOM 1177 O H2O 1177 147.356 −27.702 79.859 inf inf ATOM1178 O H2O 1178 146.074 −27.180 79.909 inf inf ATOM 1179 O H2O 1179148.034 −27.148 79.643 inf inf ATOM 1180 O H2O 1180 147.887 −26.79079.848 inf inf ATOM 1181 O H2O 1181 147.927 −26.441 79.908 inf inf ATOM1182 O H2O 1182 146.261 −26.254 79.908 inf inf ATOM 1183 O H2O 1183148.287 −26.063 79.894 inf inf ATOM 1184 O H2O 1184 148.880 −25.74279.739 inf inf ATOM 1185 O H2O 1185 148.914 −25.584 79.957 inf inf ATOM1186 O H2O 1186 149.250 −25.162 79.923 inf inf ATOM 1187 O H2O 1187145.774 −24.206 79.894 inf inf ATOM 1188 O H2O 1188 145.644 −23.89379.912 inf inf ATOM 1189 O H2O 1189 149.162 −23.453 79.936 inf inf ATOM1190 O H2O 1190 145.150 −23.307 79.904 inf inf ATOM 1191 O H2O 1191149.120 −22.748 79.903 inf inf ATOM 1192 O H2O 1192 149.115 −22.37979.903 inf inf ATOM 1193 O H2O 1193 149.050 −22.004 79.911 inf inf ATOM1194 O H2O 1194 144.786 −21.825 79.903 inf inf ATOM 1195 O H2O 1195144.404 −21.263 79.748 inf inf ATOM 1196 O H2O 1196 144.435 −20.92179.663 inf inf ATOM 1197 O H2O 1197 145.508 −21.056 79.948 inf inf ATOM1198 O H2O 1198 148.851 −21.086 79.908 inf inf ATOM 1199 O H2O 1199145.921 −20.597 79.655 inf inf ATOM 1200 O H2O 1200 146.624 −20.66379.934 inf inf ATOM 1201 O H2O 1201 148.805 −20.575 79.677 inf inf ATOM1202 O H2O 1202 147.364 −20.127 79.749 inf inf ATOM 1203 O H2O 1203148.117 −20.139 79.735 inf inf ATOM 1204 O H2O 1204 146.632 −31.60879.925 inf inf ATOM 1205 O H2O 1205 147.330 −27.507 79.967 inf inf ATOM1206 O H2O 1206 147.360 −27.173 80.053 inf inf ATOM 1207 O H2O 1207147.366 −26.809 80.072 inf inf ATOM 1208 O H2O 1208 147.001 −26.44080.108 inf inf ATOM 1209 O H2O 1209 147.001 −26.089 80.155 inf inf ATOM1210 O H2O 1210 146.620 −25.707 80.106 inf inf ATOM 1211 O H2O 1211147.736 −25.880 80.263 inf inf ATOM 1212 O H2O 1212 146.311 −25.31380.039 inf inf ATOM 1213 O H2O 1213 148.793 −25.463 80.218 inf inf ATOM1214 O H2O 1214 149.225 −24.966 80.093 inf inf ATOM 1215 O H2O 1215149.102 −24.594 80.343 inf inf ATOM 1216 O H2O 1216 149.096 −24.22580.337 inf inf ATOM 1217 O H2O 1217 149.196 −23.862 80.074 inf inf ATOM1218 O H2O 1218 145.135 −23.127 80.098 inf inf ATOM 1219 O H2O 1219145.162 −22.931 80.268 inf inf ATOM 1220 O H2O 1220 144.849 −22.04379.987 inf inf ATOM 1221 O H2O 1221 148.947 −22.038 80.215 inf inf ATOM1222 O H2O 1222 148.825 −21.839 80.254 inf inf ATOM 1223 O H2O 1223145.522 −21.455 80.275 inf inf ATOM 1224 O H2O 1224 148.543 −21.22380.202 inf inf ATOM 1225 O H2O 1225 146.236 −21.008 80.324 inf inf ATOM1226 O H2O 1226 148.492 −20.896 80.108 inf inf ATOM 1227 O H2O 1227147.370 −20.747 80.228 inf inf ATOM 1228 O H2O 1228 148.090 −20.74680.207 inf inf ATOM 1229 O H2O 1229 147.369 −25.639 80.362 inf inf ATOM1230 O H2O 1230 146.992 −25.350 80.483 inf inf ATOM 1231 O H2O 1231148.489 −25.355 80.491 inf inf ATOM 1232 O H2O 1232 146.999 −25.15280.645 inf inf ATOM 1233 O H2O 1233 148.467 −25.137 80.619 inf inf ATOM1234 O H2O 1234 146.318 −24.710 80.600 inf inf ATOM 1235 O H2O 1235145.894 −24.225 80.456 inf inf ATOM 1236 O H2O 1236 145.821 −23.81980.604 inf inf ATOM 1237 O H2O 1237 145.558 −23.461 80.437 inf inf ATOM1238 O H2O 1238 145.673 −23.136 80.695 inf inf ATOM 1239 O H2O 1239145.213 −22.748 80.418 inf inf ATOM 1240 O H2O 1240 148.880 −22.74880.480 inf inf ATOM 1241 O H2O 1241 148.877 −22.379 80.477 inf inf ATOM1242 O H2O 1242 148.632 −22.022 80.611 inf inf ATOM 1243 O H2O 1243148.288 −21.647 80.633 inf inf ATOM 1244 O H2O 1244 145.907 −21.46680.624 inf inf ATOM 1245 O H2O 1245 147.398 −21.200 80.559 inf inf ATOM1246 O H2O 1246 148.109 −21.271 80.458 inf inf ATOM 1247 O H2O 1247147.001 −21.124 80.604 inf inf ATOM 1248 O H2O 1248 148.062 −20.94980.316 inf inf ATOM 1249 O H2O 1249 148.108 −24.957 80.817 inf inf ATOM1250 O H2O 1250 146.487 −24.554 80.971 inf inf ATOM 1251 O H2O 1251148.087 −24.736 80.946 inf inf ATOM 1252 O H2O 1252 146.304 −24.37880.959 inf inf ATOM 1253 O H2O 1253 146.090 −23.851 80.999 inf inf ATOM1254 O H2O 1254 146.076 −23.487 81.012 inf inf ATOM 1255 O H2O 1255146.081 −23.117 81.006 inf inf ATOM 1256 O H2O 1256 146.081 −22.74881.003 inf inf ATOM 1257 O H2O 1257 145.568 −22.379 80.760 inf inf ATOM1258 O H2O 1258 148.489 −22.379 80.843 inf inf ATOM 1259 O H2O 1259148.106 −22.196 81.002 inf inf ATOM 1260 O H2O 1260 146.242 −21.59980.888 inf inf ATOM 1261 O H2O 1261 147.014 −21.806 81.056 inf inf ATOM1262 O H2O 1262 147.739 −21.845 80.964 inf inf ATOM 1263 O H2O 1263146.632 −21.300 80.783 inf inf ATOM 1264 O H2O 1264 147.370 −24.61981.232 inf inf ATOM 1265 O H2O 1265 146.595 −24.262 81.250 inf inf ATOM1266 O H2O 1266 147.727 −24.385 81.343 inf inf ATOM 1267 O H2O 1267146.275 −23.851 81.183 inf inf ATOM 1268 O H2O 1268 148.255 −23.83681.322 inf inf ATOM 1269 O H2O 1269 148.264 −23.487 81.336 inf inf ATOM1270 O H2O 1270 146.617 −23.298 81.405 inf inf ATOM 1271 O H2O 1271148.173 −23.069 81.308 inf inf ATOM 1272 O H2O 1272 147.002 −22.68181.331 inf inf ATOM 1273 O H2O 1273 147.751 −22.915 81.420 inf inf ATOM1274 O H2O 1274 146.632 −22.380 81.191 inf inf ATOM 1275 O H2O 1275147.728 −22.392 81.160 inf inf ATOM 1276 O H2O 1276 147.001 −22.02081.123 inf inf ATOM 1277 O H2O 1277 147.370 −24.195 81.476 inf inf ATOM1278 O H2O 1278 147.370 −23.858 81.573 inf inf ATOM 1279 O H2O 1279147.002 −23.487 81.559 inf inf ATOM 1280 O H2O 1280 147.009 −23.13481.506 inf inf TER

1-24. (canceled)
 25. A compound comprising the formula:

wherein R₁ and R₂ are hydrogen, halo, alkyl, alkenyl, ydroxyl, alkoxy,thioalkyl, thiol, phosphino, ROH, or RNH₂ group, where R is alkyl, andR₃ is alkyl, alkenyl, aryl, aralkyl, ROH, or RNH₂ group, where R isalkyl, and X and Y are independently O or S, or a pharmaceuticallyacceptable salt thereof.
 26. The compound of claim 25, wherein R₁ isalkyl, alkenyl, ROH, or RNH₂.
 27. The compound of claim 25, wherein R₁is methyl, ethyl, or isopropyl.
 28. The compound of claim 25, wherein R₂is halo, alkyl, or C1-C3 alkoxy.
 29. The compound of claim 25, wherein Xis S.
 30. The compound of claim 25, wherein X and Y are each S.
 31. Thecompound of claim 25, wherein R₃ is C1-C3 alkyl. 32-62. (canceled)
 63. Amethod for treating a subject infected with a retrovirus, the methodcomprising administering to the subject an effective antiviral dose ofany one of the compounds of claim
 25. 64. A method for killing HIV virusin a cell, the method comprising administering to the cell an effectiveanti-viral amount of any one of the compounds of claim
 25. 65. A methodfor inhibiting the growth of HIV in a cell, the method comprisingadministering to the cell an effective inhibitory dose of any of thecompounds of claim
 25. 66-130. (canceled)